Production of human hemoglobin in transgenic pigs

ABSTRACT

The present invention relates to the use of transgenic pigs for the production of human hemoglobin. The transgenic pigs of the invention may be used as an efficient and economical source of cell-free human hemoglobin that may be used for transfusions and other medical applications in humans.

This application is a continuation of U.S. application Ser. No.08/105,989, filed Aug. 11, 1993, now U.S. Pat. No. 5,922,854, which is acontinuation in-part of U.S. application Ser. No. 08/030,897, filed Mar.15, 1993, now abandoned which is a continuation-in-part of U.S. Ser. No.07/897,648, filed Jun. 12, 1992, now abandoned which is acontinuation-in-part of U.S. application Ser. No. 07/717,774, filed Jun.14, 1991 now abandoned.

INTRODUCTION

The present invention relates to the use of transgenic pigs for theproduction of human hemoglobin. The transgenic pigs of the invention maybe used as an efficient and economical source of cell-free humanhemoglobin that may be used for transfusions and other medicalapplications in humans.

BACKGROUND OF THE INVENTION HEMOGLOBIN

Oxygen absorbed through the lungs is carried by hemoglobin in red bloodcells for delivery to tissues throughout the body. At high oxygentensions, such as those found in the proximity of the lungs, oxygenbinds to hemoglobin, but is released in areas of low oxygen tension,where it is needed.

Each hemoglobin molecule consists of two alpha globin and two betaglobin subunits. Each subunit, in turn, is noncovalently associated withan iron-containing heme group capable of carrying an oxygen molecule.Thus, each hemoglobin tetramer is capable of binding four molecules ofoxygen. The subunits work together in switching between twoconformational states to facilitate uptake and release of oxygen at thelungs and tissues, respectively. This effect is commonly referred to asheme-heme interaction or cooperativity.

The hemoglobins of many animals are able to interact with biologiceffector molecules that can further enhance oxygen binding and release.This enhancement is manifested in changes which affect the allostericequilibrium between the two conformational states of hemoglobin. Forexample, human and pig hemoglobin can bind 2, 3 diphosphoglycerate (2,3DPG), which influences the equilibrium between the two conformationalstates of the tetramer and has the net effect of lowering the overallaffinity for oxygen at the tissue level. As a result, 2,3-DPG increasesthe efficiency of oxygen delivery to the tissues.

GLOBIN GENE EXPRESSION

Hemoglobin protein is expressed in a tissue specific manner in red bloodcells where it accounts for approximately ninety percent of totalcellular protein. Thus, red blood cells, which have lost their nucleusand all but a minimal number of organelles, are effectivelymembrane-enclosed packets of hemoglobin dedicated to oxygen transfer.

Humans and various other species produce different types of hemoglobinduring embryonic, fetal, and adult developmental periods. Therefore, thefactors that influence globin gene expression must be able to achievetissue specific control, quantitative control, and developmentallyregulated control of globin expression.

Human globin genes are found in clusters on chromosome 16 for alpha (α)globin and chromosome 11 for beta (β) globin. The human beta globin genecluster consists of about 50 kb of DNA that includes one embryonic geneencoding epsilon (ε) globin, two fetal genes encoding gamma (γ) G andgamma A globin, and two adult genes encoding delta (δ) and beta (β)globin, in that order (Fritsch et al., 1980, Cell 19:959-972).

It has been found that DNA sequences both upstream and downstream of theβ globin translation initiation site are involved in the regulation of βglobin gene expression (Wright et al., 1984, Cell 38:263). Inparticular, a series of four Dnase I super hypersensitive sites (nowreferred to as the locus control region, or LCR) located about 50kilobases upstream of the human beta globin gene are extremely importantin eliciting properly regulated beta globin-locus expression (Tuan etal., 1985, Proc. Natl. Acad. Sci. U.S.A. 83:1359-1363; PCT PatentApplication WO 8901517 by Grosveld; Behringer et al., 1989, Science245:971-973; Enver et al., 1989, Proc. Natl. Acad. Sci. U.S.A.86:7033-7037; Hanscombe et al., 1989, Genes Dev. 3:1572-1581; VanAssendelft et al., 1989, Cell 56:967-977; Grosveld et al., 1987, Cell51:975-985).

THE NEED FOR A BLOOD SUBSTITUTE

Recently, the molecular aspects of globin gene expression have met witheven greater interest as researchers have attempted to use geneticengineering to produce a synthetic blood that would avoid the pitfallsof donor generated blood. In 1988, between 12 million and 14 millionunits of blood were used in the United States alone (Andrews, Feb. 18,1990, New York Times), an enormous volume precariously dependent onvolunteer blood donations. About 5 percent of donated blood is infectedby hepatitis virus (Id.) and, although screening procedures for HIVinfection are generally effective, the prospect of contractingtransfusion related A.I.D.S. remains a much feared possibility.Furthermore, transfused blood must be compatible with the blood type ofthe transfusion recipient; the donated blood supply may be unable toprovide transfusions to individuals with rare blood types. In contrast,hemoglobin produced by genetic engineering would not require blood typematching, would be virus-free, and would be available in potentiallyunlimited amounts. Several research groups have explored the possibilityof expressing hemoglobin in microorganisms. For example, seeInternational Application No. PCT/US88/01534 by Hoffman and Nagai, whichpresents, in working examples, production of human globin protein in E.coli.

TRANSGENIC ANIMALS

A transgenic animal is a non-human animal containing at least oneforeign gene, called a transgene, in its genetic material. Preferably,the transgene is contained in the animal's germ line such that it can betransmitted to the animal's offspring. A number of techniques may beused to introduce the transgene into an animal's genetic material,including, but not limited to, microinjection of the transgene intopronuclei of fertilized eggs and manipulation of embryonic stem cells(U.S. Pat. No. 4,873,191 by Wagner and Hoppe; Palmiter and Brinster,1986, Ann. Rev. Genet. 20:465-499; French Patent Application 2593827published Aug. 7, 1987). Transgenic animals may carry the transgene inall their cells or may be genetically mosaic.

Although the majority of studies have involved transgenic mice, otherspecies of transgenic animal have also been produced, such as rabbits,sheep, pigs (Hammer et al., 1985, Nature 315:680-683) and chickens(Salter et al., 1987, Virology 157:236-240). Transgenic animals arecurrently being developed to serve as bioreactors for the production ofuseful pharmaceutical compounds (Van Brunt, 1988, Bio/Technology6:1149-1154; Wilmut et al., 1988, New Scientist (July 7 issue) pp.56-59).

Methods of expressing recombinant protein via transgenic livestock havean important theoretical advantage over protein production inrecombinant bacteria and yeast; namely, the ability to produce large,complex proteins in which post-translational modifications, includingglycosylation, phosphorylation, subunit assembly, etc. are critical forthe activity of the molecule.

In practice, however, the creation of transgenic livestock has provedproblematic. Not only is it technically difficult to produce transgenicembryos, but mature transgenic animals that produce significantquantities of recombinant protein may prove inviable. In pigs inparticular, the experience has been that pigs carrying a growth hormoneencoding transgene (the only transgene introduced into pigs prior to thepresent invention) suffered from a number of health problems, includingsevere arthritis, lack of coordination in their rear legs,susceptibility to stress, anoestrus in gilts and lack of libido in boars(Wilmut et al., supra). This is in contrast to transgenic mice carryinga growth hormone transgene, which appeared to be healthy (Palmiter etal., 1982, Nature 300:611-615). Thus, prior to the present invention,healthy transgenic pigs (which efficiently express their transgene(s))had not been produced.

EXPRESSION OF GLOBIN GENES IN TRANSGENIC ANIMALS

Transgenic mice carrying human globin transgenes have been used instudying the molecular biology of globin gene expression. A hybridmouse/human adult beta globin gene was described by Magram et al. in1985 (Nature 315:338-340). Kollias et al. then reported regulatedexpression of human gamma-A, beta, and hybrid beta/gamma globin genes intransgenic mice (1986, Cell 46:89-94). Transgenic mice expressing humanfetal gamma globin were studied by Enver et al. (1989, Proc. Natl. Acad.Sci. U.S.A. 86:7033-7037) and Constantoulakis et al. (1991, Blood77:1326-1333). Autonomous developmental control of human embryonicglobin gene switching in transgenic mice was observed by Raich et al.(1990, Science 250:1147-1149).

Transgenic mouse models for a variety of disorders of hemoglobin orhemoglobin expression have been developed, including sickle cell disease(Rubin et al., 1988, Am. J. Human Genet. 42:585-591; Greaves et al.,1990, Nature 343:183-185; Ryan et al., 1990, Science 247:566-568; Rubinet al., 1991, J. Clin. Invest. 87:639-647); thalassemia (Anderson etal., 1985, Ann. New York Acad. Sci. (USA) 445:445-451; Sorenson et al.,1990, Blood 75:1333-1336); and hereditary persistence of fetalhemoglobin (Tanaka et al., 1990, Ann. New York Acad. Sci. (USA)612:167-178).

Concurrent expression of human alpha and beta globin has led to theproduction of human hemoglobin in transgenic mice (Behringer et al.,1989, Science 245:971-973; Townes et al., 1989, Prog. Clin. Biol. Res.316A:47-61; Hanscombe et al., 1989, Genes Dev. 3:1572-1581). It wasobserved by Hanscombe et al. (supra) that transgenic fetuses with highcopy numbers of a transgene encoding alpha but not beta globin exhibitedsevere anemia and died prior to birth. Using a construct with both humanalpha and beta globin genes under the control of the beta globin LCR,live mice with low copy numbers were obtained (Id.). Metabolic labelingexperiments showed balanced mouse globin synthesis, but imbalanced humanglobin synthesis, with an alpha/beta biosynthetic ratio of about 0.6(Id.).

SUMMARY OF THE INVENTION

The present invention relates to the use of transgenic pigs for theproduction of human hemoglobin and/or human globin. It is based, atleast in part, on the discovery that transgenic pigs may be generatedthat express human hemoglobin in their erythrocytes and are healthy,suffering no deleterious effects as a result of heterologous hemoglobinproduction.

In particular embodiments, the present invention provides for transgenicpigs that express human globin genes. Such animals may be used as aparticularly efficient and economical source of human hemoglobin, inlight of (i) the relatively short periods of gestation and sexualmaturation in pigs; (ii) the size and frequency of litters, (iii) therelatively large size of the pig which provides proportionately largeyields of hemoglobin; and (iv) functional similarities between pig andhuman hemoglobins in the regulation of oxygen binding affinity whichenables the transgenic pigs to remain healthy in the presence of highlevels of human hemoglobin.

The present invention also provides for recombinant nucleic acidconstructs that may be used to generate transgenic pigs. In specific,nonlimiting embodiments, such constructs (1) place the human alpha andbeta globin genes under the same promoter; (ii) comprise the pig adultbeta globin gene regulatory region, comprising the promoter or the 3'region of the pig beta globin gene; and/or (iii) comprise the humanglobin genes under the control of the porcine locus control region(LCR).

The present invention also provides for constructs comprising anoptimized human β-globin gene in which said human β-globin gene isgenetically engineered to be similar to the pig β-globin gene, butwithout altering the amino acid sequence of the encoded wild-type humanβ-globin. Such constructs may increase the level of human β-globin intransgenic pigs by affecting mRNA structure, stability or rate oftranslation.

In an additional embodiment, the present invention provides for a hybridhemoglobin that comprises human α globin and pig β globin. The wholeblood from transgenic pigs expressing this hybrid hemoglobin appears toexhibit a P₅₀ that is advantageously higher than that of native human orpig blood.

The present invention also provides for a method of producing humanhemoglobin comprising (i) introducing a human alpha globin and a humanbeta globin gene, under the control of a suitable promoter or promoters,into the genetic material of a pig so as to create a transgenic pig thatexpresses human hemoglobin in at least some of its red blood cells; (ii)collecting red blood cells from the transgenic pig; (iii) releasing thecontents of the collected red blood cells; and (iv) subjecting thereleased contents of the red blood cells to a purification procedurethat substantially separates human hemoglobin from pig hemoglobin. In apreferred embodiment of the invention, human hemoglobin may be separatedfrom pig hemoglobin by DEAE anion exchange column chromatography.

DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, 1O, 1P,1Q, 1R, 1S, 1T, 1U, 1V, 1W, 1X, and 1Y. Recombinant nucleic acidconstructs.

FIG. 1A. Construct ααβ (the "116 construct); FIG. 1B. Construct αpβ (the"185" construct); FIG. 1C. Construct βpα (the "290" construct); FIG. 1D.Construct εpζβα; FIG. 1E. Construct ζpεαpβ; FIG. 1F. Construct αpβcarrying a β108 Asn→Asp mutation (the "hemoglobin Yoshizuka construct");FIG. 1G. Construct αpβ carrying a β108 Asn→Lys mutation (the "hemoglobinPresbyterian construct"); FIG. 1H. Construct αpβ(Δα) coinjected with LCRα (the "285" construct); FIG. 1I. Construct αpβ carrying an α134 Thr→Cysmutation (the "227" construct); FIG. 1J. Construct αpβ carrying an α104Cys→Ser mutation (the "227" construct), a β93 Cys→Ala mutation, and aβ112 Cys→Val mutation (the "228" construct); FIG. 1K. Construct αpδ (the"263" construct); and FIG. 1L. Construct αpδ (Δα) coinjected with LCR α(the "274" construct); FIG. 1M. Construct LCR α coinjected with LCR εβ(the "240" construct); FIG. 1N. Construct αpβ carrying a β61 Lys→Metmutation (the "Hemoglobin Bologna" construct); FIG. 1O. Construct LCRεαβ (the "318" construct); FIG. 1P. Construct LCR αεβ (the "319"construct); FIG. 1Q. Construct LCR ααεβ (the "329" construct); FIG. 1R.Construct LCR αε(^(pig) βp)β (the "339" construct); FIG. 1S. Constructαpβ carrying an α75 Asp→Cys mutation (the "340" construct); FIG. 1T.Construct αpβ carrying an α42 Tyr→Arg mutation (the "341" construct);FIG. 1U. Construct LCR εβαα (the "343" construct); FIG. 1V. ConstructLCR εβα (the "347" construct); FIG. 1W. Construct αpβ carrying an α42Tyr→Lys mutation; FIG. 1X. Construct αpβ carrying an α42 Tyr→Argmutation; and a β99 Asp→Glu mutation; FIG. 1Y. Construct αpβ carrying anα42 Tyr→Lys mutation; and a β99 Asp→Glu mutation.

FIG. 2. Transgenic pig.

FIGS. 3A and 3B. Demonstration of human hemoglobin expression intransgenic pigs. FIG. 3A. Isoelectric focusing gel analysis. FIG. 3B.Triton-acid urea gel of hemolysates of red blood cells representinghuman blood (lane 1); blood from transgenic pig 12-1 (lane 2), 9-3 (lane3), and 6-3 (lane 4); and pig blood (lane 5) shows under-expression ofhuman β globin relative to human α globin in the transgenic animals.

FIGS. 4A, 4B, 4C and 4D. Separation of human hemoglobin and pighemoglobin by DEAE chromatography. FIG. 4A. Hemolyzed mixture of humanand pig red blood cells; FIG. 4B. Hemolysate of red blood cellscollected from transgenic pig 6-3. FIG. 4C. Human and mouse hemoglobindo not separate by DEAE chromatography under these conditions. FIG. 4D.Isoelectric focusing of human hemoglobin purified from pig hemoglobin.

FIG. 5. Isoelectric focussing gel of reassociated pig hemoglobin (lane1); reassociated pig/human hemoglobin mixture (lanes 2 and 4);reassociated human hemoglobin (lane 3); and transgenic pig hemoglobin(lane 5).

FIG. 6. Separation of human hemoglobin by QCPI chromatography.

FIG. 7. Oxygen affinity of transgenic hemoglobin.

FIG. 8. DNA sequence (SEQ. ID NO:1) of the pig adult beta globin generegulatory region, including the promoter region. Sequence extending to869 base pairs upstream of the ATG initiator codon (boxed) of the pigbeta globin gene is shown. The position of the initiation of mRNA, thecap site, is indicated by an arrow. The sequences corresponding to GATAtranscription factor binding sites are underlined.

FIG. 9. Comparison of pig (SEQ. ID NO: 1) (top) and human (SEQ ID NO's:2 & 3) (bottom) beta globin regulatory sequences. Differences in the twosequences are marked by asterisks.

FIG. 10. Graph depicting the percent homology between pig and humanadult beta globin gene regulatory sequences, with base pair distancefrom the initiator codon mapped on the abscissa. A comparison of mouseand human sequences is also shown (dotted line with error bar).

FIG. 11. Map of plasmid pgem5/PigβPr(k) which contains the DNA sequencedepicted in FIG. 8.

FIG. 12. Representation of the 339 and 354 cassettes for the productionof human hemoglobin in transgenic pigs.

FIG. 13. Map of plasmid pSaf/Pigε(k), containing the pig ε gene.

FIG. 14. Representation of the 426 and 427 expression cassettes for theproduction of ε^(pig) β^(human) and α^(human) hemoglobins in transgenicpigs.

FIG. 15. Iso-electric focussing gel of hemoglobin produced by transgenicpig 70-3, which carries the 339 construct, and by transgenic pig 6-3,which carries the 116 construct. Human hemoglobin is run as a standard.

FIG. 16. Map of plasmid pig 3'β containing the 3' end of the pig betaglobin gene.

FIG. 17. Transgenic pigs obtained from construct "339" (See FIG. 1R).Levels of human hemoglobin expression and copy number are shown.

FIG. 18. Isoelectric focussing gel of hemoglobin levels in transgenicpigs obtained using construct "339".

FIG. 19. Isoelectric focussing gel demonstrating levels of hemoglobinexpression in representative transgene positive 38-4 offspring carryingthe "185" construct (or αpβ construct; see FIG. 1B).

FIG. 20. Molecular modeling of hybrid human α/pig β and human α/human βhemoglobin molecules. β subunits are in blue, α subunits in red. Abovethe middle helix of the β human (blue) one can see a gap in the greencontour (see arrow). In the hybrid this gap is filed in. This differenceis due to a change at β112 Cys→Val where Valine contributes to greaterhydrophobic interactions.

FIG. 21. Molecular modeling demonstrating the differences at the α₁ β₁interface between a β globin containing Cys at position 112 (the yellowmolecule) and a β globin with Val at position 112 (the white molecule).Cys is yellow, Val is white and the opposing α interface is red. Val isflexible. One arm of its branch can easily move for a nearly perfect fitagainst the α subunit residues. The yellow Cys is slightly furtherallowing for a small gap (see arrow). Biosyn's standard default Van derWaal's distance was used.

FIG. 22. Purification of Hb Presbyterian from transgenic pig hemosylate.

FIG. 23. Characterization of purified Hb Presbyterian by HPLC showingseparation of the heme moiety, pig α globin ("p alpha"), human betaglobin ("h beta"), human alpha globin ("h alpha") and pig beta globin("p beta").

FIG. 24. Oxygen binding curve for Hb Presbyterian.

FIG. 25. Purification of Hb Yoshizuka from transgenic pig hemolysate.

FIGS. 26A and 26B. Porcine β LCR clones. FIG. 26A Restriction analysisof lambda phage clone Phage L and Phage H. The insert shows the mostprobable arrangement of porcine β globin genes. The location of theprobe used to screen the library is shown. FIG. 26B Comparison of thedistances of human LCRs from human ξ genes with porcine LCRs fromporcine ξ genes.

FIG. 27.(A) PH1-TA1 (SEQ ID NO: 4): Sequence of 3' end of the plasmidPH1end. This is part of porcine LCR I. (B) Comparison of PH1-TA1 withhuman β-globin region on chromosome 11 (SEQ ID NO: 5). The humansequence (from 12499-12901) is part of LCR I.

FIG. 28. Joined plcr2 (SEQ ID NO: 6): The 477 bp sequence of 5' end ofplasmid PH1 was joined with 534 bp sequence of 3' end of plasmid PH2.This is part of porcine LCR II.

FIG. 29. Comparison of joined plcr2 with human β-globin region onchromosome 11. The human sequence (from 7276-8017) is part of LCR II.

FIGS. 30A and 30B. PH2-T7. FIG. 30A Sequence of 540 end of plasmidPH2-T7. FIG. 30B. Comparison of PH2-T7 with human β-globin region onchromosome 11. The human sequence (from 1450-1487) is part of LCR III.

FIG. 31. Schematic of optimized β-globin gene including importantrestriction sites used for construction. Promoter region, Interveningsequences I and II (IVSI, IVSII) as well as poly-A and 3'UTR region arepig sequences. Exon 1, 2 and 3 encode human β-globin with codonsoptimized for use in the pig system.

FIG. 32. Comparison of coding sequences of optimized, human and pigβ-globin genes showing percent homology between the optimized and humansequences and the human and pig sequences. Lines in the optimizedsequence indicate codon changes from the human sequence.

FIG. 33. Construct 505. This construct contains the human locus controlregion (LCR), the human α-globin gene driven by its own promoter, thehuman ξ-globin gene also driven by its own promoter, and the optimizedβ-globin gene which has the optimized coding region, includes theporcine introns, poly A and 3'UTR and is driven by the porcine promoter.The gene order in this construct is LCRαξβ* (where * signifies optimizedβ gene).

FIG. 34. Construct 515. This construct contains the human locus controlregion, the human α-globin gene driven by its own promoter, the humanξ-gene also driven by its own promoter, and the optimized β-globin genewhich has the optimized coding region, includes the porcine introns,poly A and 3'UTR and is driven by the porcine promoter. The gene orderin this construct is LCRξβαα (where * signifies optimized β gene).

FIG. 35. Comparison of human and pig β-globin coding sequences. Thefigure is divided into Exons 1, 2 and 3. Differences are signified bysmall letters in the pig (SEQ. ID NO's: 15, 16 & 17) (bottom) sequence.Codons with changes are underlined.

FIG. 36. Comparison of human (SEQ ID NO's: 12, 13 & 14) and optimized(SEQ ID NO's: 18, 20 & 22) β-globin coding sequences. The figure isdivided into Exons 1, 2 and 3. Differences are signified by smallletters in the optimized (bottom) sequence. Codons with changes areunderlined.

FIG 37. Comparison of optimized (SEQ ID NO's: 18, 20 & 22) and pig (SEQ.ID NO's: 15, 16 & 17) β-globin coding sequences. Figure is divided intoExons 1, 2 and 3. Differences are signified by small letters in the pig(bottom) sequence. Codons with changes are underlined.

FIG. 38. Coding sequences (SEQ. ID NO's: 18, 20 & 22) and amino acidsequence (SEQ. ID NO's: 19, 21 & 23) of optimized β-globin gene. Threedashes are placed between Exons.

FIG. 39. Comparison of human (SEQ. ID NO's: 24, 25 & 26) and optimized(SEQ ID NO's: 19, 21 & 23) β-globin amino acid sequence indicating thatthey are identical.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for a method of producing humanhemoglobin that utilizes transgenic pigs, novel globin-encoding nucleicacid constructs, and transgenic pigs that express human hemoglobin. Forpurposes of clarity of description, and not by way of limitation, thedetailed description of the invention is divided into the followingsubsections:

(i) preparation of globin gene constructs;

(ii) preparation of transgenic pigs;

(iii) preparation of human hemoglobin and its separation from pighemoglobin; and

(iv) preparation of human/pig hybrid hemoglobin.

Preparation of Globin Gene Constructs

The present invention provides for a method of producing human globinand/or hemoglobin in transgenic pigs. Human hemoglobin is defined hereinto refer to hemoglobin formed by globin chains encoded by human globingenes (including alpha, beta, delta, gamma, epsilon and zeta genes) orvariants thereof which are naturally occurring or the products ofgenetic engineering. Such variants are at least about ninety percenthomologous in amino acid sequence to a naturally occurring humanhemoglobin. In preferred embodiments, the human hemoglobin of theinvention comprises a human alpha globin and a human beta globin chain.The human hemoglobin of the invention comprises at least two differentglobin chains, but may comprise more than two chains, to form, forexample, a tetrameric molecule, octameric molecule, etc. In preferredembodiments of the invention, human hemoglobin consists of two humanalpha globin chains and two human beta globin chains. As discussedinfra, the present invention also provides for hybrid hemoglobinscomprising human α globin and pig β globin.

According to particular embodiments of the present invention, at leastone human globin gene, such as a human alpha and/or a human beta globingene, under the control of a suitable promoter or promoters, is insertedinto the genetic material of a pig so as to create a transgenic pig thatcarries human globin in at least some of its red blood cells. Thisrequires the preparation of appropriate recombinant nucleic acidsequences. In preferred embodiments of the invention, both human α andhuman β genes are expressed. In an alternative embodiment, only human αglobin or human β globin is expressed. In further embodiments, humanembryonic or fetal globin genes are expressed or are used asdevelopmental expression regulators of adult genes.

Human alpha and beta globin genes may be obtained from publiclyavailable clones, e.g. as described in Swanson et al., 1992,Bio/Technol. 10:557-559. Nucleic acid sequences encoding human alpha andbeta globin proteins may be introduced into an animal via two differentspecies of recombinant constructs, one which encodes human alpha globin,the other encoding human beta globin; alternatively, and preferably,both alpha and beta-encoding sequences may be comprised in the samerecombinant construct. The pig epsilon globin gene is contained inplasmid psaf/pig ε (k) (FIG. 13), deposited with the ATCC and assignedaccession number 75373.

A suitable promoter, according to the invention, is a promoter which candirect transcription of human alpha and/or beta globin genes in redblood cells. Such a promoter is preferably selectively active inerythroid cells. This would include, but is not limited to, a globingene promoter, such as the human alpha, beta, delta, epsilon or zetapromoters, or a globin promoter from another species. It may, forexample, be useful to utilize pig globin promoter sequences. Forexample, as discussed in Section 10, infra, the use of the endogenouspig β globin gene control region, as contained in plasmidPgem5/Pigβpr(K), deposited with the ATCC and assigned accession number75371 and having the sequence set forth in FIG. 8, has been shown tooperate particularly efficiently. The human alpha and beta globin genesmay be placed under the control of different promoters, but, since ithas been inferred that vastly different levels of globin chainproduction may result in lethality, it may be preferable to place thehuman alpha and beta globin genes under the control of the same promotersequence. In order to avoid chain imbalance and/or titration oftranscription factors due to constitutive β-globin promoter activity inan inappropriate cell type, it is desirable to design a construct whichleads to coordinate expression of human alpha and beta globin genes atthe same time in development and at quantitatively similar levels.

In one particular, non-limiting embodiment of the invention, a constructcomprising the ααβ construct (also termed the "116" construct; Swansonet al., 1992, Bio/Technol. 10:557-559; see FIG. 1A) may be utilized.Although this construct, when present as a transgene at high copynumber, has resulted in deleterious effects in mice, it has been used toproduce healthy transgenic pigs (see Example Section 6, infra).

In another particular, non-limiting embodiment of the invention, aconstruct comprising the αpβ sequence (also termed the "185" construct;see FIG. 1B) may be used. Such a construct has the advantage of placingboth alpha and beta globin-encoding sequences under the control of thesame promoter (the alpha globin promoter).

In another particular, non-limiting embodiment of the invention, aconstruct coding for di-alpha globin like polypeptides may be introducedto form transgenic pigs that produce human hemoglobins with decreaseddimerization and an increased half-life (WO Patent 9013645).

In yet another particular, non-limiting embodiment of the invention, aconstruct comprising the human adult alpha globin and epsilon globingene, the pig beta globin gene control region and the human beta globingene (the "339 construct, see FIG. 1R) may be used.

Furthermore, the incorporation of a human or pig epsilon globin geneinto the construct may facilitate the production of high hemoglobinlevels. The pig epsilon globin gene may permit correct developmentalregulation of the adult β globin gene. High levels of expression ofintroduced adult alpha globin gene(s) may result in a chain imbalanceproblem during intrauterine development of a transgenic pig embryo(because an adult beta globin gene in the construct would not yet beexpressed) thereby compromising the viability of the embryo. Byproviding high levels of embryonic globins during development, theviability of such embryos may be improved. The pig epsilon globin gene,as contained in plasmid Psaf/Pigε, deposited with the ATCC and assignedaccession number 75373, is shown in FIG. 13.

The present invention, in further specific embodiments, provides for (i)the construct βpα, in which the human alpha and beta globin genes aredriven by separate copies of the human beta globin promoter (FIG. 1C);(ii) the εpζβpα construct, which comprises human embryonic genes zetaand epsilon under the control of the epsilon promoter and both alpha andbeta genes under the control of the beta promoter (FIG. 1D); (iii) theζpεαpβ construct, which comprises human embryonic genes zeta and epsilonunder the control of the zeta promoter and both alpha and beta genesunder the control of the alpha promoter (FIG. 1E); (iv) the αpβconstruct carrying a mutation that results in an aspartic acid residue(rather than an asparagine residue) at amino acid number 108 of β globinprotein, to produce hemoglobin Yoshizuka (FIG. 1F, construct "294"); (v)the αpβ construct carrying a mutation that results in a lysine residue(rather than an asparagine residue) at amino acid number 108 of β-globinprotein, to produce hemoglobin Presbyterian (FIG. 1G, construct "293");(vi) the αpβ(Δα) construct, coinjected with LCR α which comprises thehuman β-globin gene under the control of the human α-globin promoter anda separate nucleic acid fragment comprising the human α-globin geneunder its own promoter (FIG. 1H); (vii) the αpβ construct carrying amutation that results in a cysteine residue (rather than a threonineresidue) at amino acid number 134 of α-globin protein (FIG. 1I); (viii)the αpβ construct carrying a mutation that results in a serine residue(rather than a cysteine residue) at amino acid number 104 of theα-globin protein, an alanine residue (rather than a cysteine residue) atamino acid number 93 of the β-globin protein and a valine residue(rather than a cysteine residue) at amino acid number 112 of theβ-globin protein (FIG. 1J); (ix) the αpδ construct, which comprises thehuman adult α-globin promoter under its own promoter and the humanδ-globin gene under the control of the human adult α-globin promoter(FIG. 1K); (x) Construct αpδ(Δα) coinjected with LCR α, which comprisesthe human δ-globin gene under the control of the human α-globin promoterand a separate nucleic acid fragment comprising the human α-globin geneunder its own promoter (FIG. 1L); (xi) Construct LCR α coinjected withLCR εβ, which comprises the human α-globin gene under the control of itsown promoter and a separate nucleic acid fragment comprising the humanembryonic ε-globin gene and the adult β-globin gene under the control oftheir own promoters (FIG. 1M); (xii) the αpβ construct carrying amutation that results in a methionine residue (rather than a lysineresidue) at amino acid number 61 of the α-globin protein (FIG. 1N);(xiii) the εαβ construct, which comprises the human embryonic epsilongene, the human adult alpha globin gene and the human adult beta globingene linked in tandem from 5'- to 3' (FIG. 1O); (xiv) the αεβ construct,which comprises the human adult alpha-globin gene, the human embryonicepsilon globin gene and the human adult beta globin gene linked intandem from 5'- to 3' (FIG. 1P); (xv) the ααεβ construct, whichcomprises two copies of the human adult alpha-globin gene, the humanembryonic epsilon globin gene and the human adult beta globin genelinked in tandem from 5'- to 3' (FIG. 1Q); (xvi) the αε (^(pig) βp)βconstruct, which comprises the human adult alpha-globin gene, the humanembryonic epsilon globin gene and the human adult beta globin gene underthe control of the endogenous porcine adult beta globin promoter alllinked in tandem from 5'- to 3' (FIG. 1R); (xvii) the αpβ constructcarrying a mutation that results in a cysteine residue (rather than anaspartic acid residue) at amino acid number 75 of the α-globin protein(FIG. 1S); (xviii) the αpβ construct carrying a mutation that results inan arginine residue (rather than a tyrosine residue) at amino acidnumber 42 at the α-globin protein (FIG. 1T); (xvix) the LCR εβααconstruct, which comprises the human embryonic epsilon globin gene, thehuman adult beta globin gene and two copies of the human adultalpha-globin gene linked in tandem from 5'- to 3' (FIG. 1U); (xx) theLCR εβα construct, which comprises the human embryonic epsilon globingene, the human adult beta globin gene and the human adult alpha-globingene linked in tandem from 5'- to 3' (FIG. 1V); (xxi) the αpβ constructcarrying a mutation that results in a lysine residue (rather than atyrosine residue) at amino acid number 42 of the α-globin protein (FIG.1W); (xxii) the αpβ construct carrying a mutation that results in anarginine residue (rather than a tyrosine residue) at amino acid number42 at the α-globin protein and a glutamic acid residue (rather than anaspartic acid residue) at amino acid number 99 of the β-globin protein(FIG. 1X); (xxiii) the αpβ construct carrying a mutation that results ina lysine residue (rather than a tyrosine residue) at amino acid number42 of the α-globin protein and a glutamic acid residue (rather than anaspartic acid residue) at amino acid number 99 of the β-globin protein(FIG. 1Y); and (xxiv) the α^(pig) ε(^(pig) βp)β construct comprising thepig epsilon globin gene and beta globin control region (constructs 426and 427, FIG. 14).

In transgenic pigs expressing human hemoglobin three types of hemoglobindimers are detectable: pig α/pig β, human α/human β, and hybrid humanα/pig β. In certain embodiments of the invention, it may be desirable todecrease the amount of hybrid hemoglobin. Accordingly, the molecularbasis for the formation of hybrid hemoglobin has been investigated usingmolecular modeling studies. Based on the information derived from thesestudies, the human alpha and beta globin structures can be modified toincrease the level of human α/human β dimers (See Section 11.), so thatin further embodiments of the invention, constructs comprising the αpβsequence may be modified to code for α or β globin proteins carryingamino acid changes that will lead to increases in the level of humanα/human β hemoglobin dimers in transgenic pigs. The present invention,provides for constructs which encode human α globin and human β globincarrying one or more of the following mutations in the a globinmolecule: (1) a Thr at position 30 instead of Glu; (ii) a Tyr atposition 36 instead of Phe; (iii) a Phe instead of Leu at position 106;(iv) a Ser or Cys instead of Val at position 107; and/or (v) a Cysinstead of Ala at position 111. In specific embodiments, the constructcarrying such mutation(s) is the αpβ construct. The present invention,in further embodiments, provides for constructs which encode human αglobin and human β globin carrying one or more of the followingmutations in the β globin molecule: (1) a Leu instead of Val at position33; (ii) a Val or Ile instead of Cys at position 112; (iii) a Val or Leuinstead of Ala at position at position 115; (iv) a His instead of Gly atposition 119; (v) a Met instead of Pro at position 125; (vi) an Ileinstead of Ala at position 128; and/or (vii) a Glu instead of Gln atposition 131; and/or (viii) a Glu instead of Gln at position 131. Inspecific embodiments, the construct carrying the mutation(s) is the αpβconstruct.

In further embodiments it may be desirable to modify the human β-globingene to optimize expression in transgenic pigs. For example, the humanβ-globin gene, from the promoter region through the coding sequence andinto the polyadenylation site and 3' untranslated region, may beengineered to be similar to the pig β-globin gene, but without alteringthe amino acid sequence from that of the authentic wild-type humanβ-globin. Such an optimized gene is contained in the plasmid designatedpGEM3 β* Δ3', deposited with the American Type Culture Collection (ATCC)and assigned accession number 75520. Constructs which contain theoptimized human β-globin gene, may be used to increase the levels ofβ-globin expressed in transgenic animals (constructs 505 and 515, FIGS.34 and 35 respectively).

In further embodiments the porcine LCR region as depicted in FIG. 26Aand contained in plasmids designated pPH1 and pPH2 (deposited with theATCC and assigned accession numbers 75518 and 75519), may be used inplasmid constructs to enhance the expression of globin proteins intransgenic pigs. The porcine LCR may also be useful in the expression ofnon-globin proteins in pig erythrocytes.

In further embodiments it may be desirable to include, in constructs,the untranslated 3' end of the pig beta globin gene as contained inplasmid pPig3'β (FIG. 16) as deposited with the ATCC and assignedaccession number 75372. (see, for example, construct 354 in FIG. 12 andFIGS. 426 and 427 in FIG. 14). Such constructs may also be useful in theexpression of non-globin protein in pig erythrocytes.

In further embodiments, the pig beta globin control region depicted inFIGS. 8 and 9 may be used in constructs that encode non-globin proteinsfor the expression of said proteins in transgenic pig or other non-humanerythrocytes.

The recombinant nucleic acid constructs described above may be insertedinto any suitable plasmid, bacteriophage, or viral vector foramplification, and may thereby be propagated using methods known in theart, such as those described in Maniatis et al., 1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y. In the workingexamples presented below, the pUC vector (Yanish-Perron et al., 1985,Gene 103-119) was utilized.

The present invention further provides for isolated and purified nucleicacids comprising the pig adult beta globin promoter regulatory region,the pig 3' beta globin region, and the pig epsilon globin gene ascomprised, respectively, in plasmids pGem5/Pigβpr(K) (ATCC accession no.75371), pPig3'β (ATCC accession no. 75372), and Psaf/pigε(k) (ATCCaccession no. 75373), respectively.

Constructs may desirably be linearized for preparation of transgenicpigs. Vector sequence may desirably be removed.

Preparation of Transgenic Pigs

The recombinant constructs described above may be used to produce atransgenic pig by any method known in the art, including but not limitedto, microinjection, embryonic stem (ES) cell manipulation,electroporation, cell gun, transfection, transduction, retroviralinfection, etc. Species of constructs may be introduced individually orin groups of two or more types of construct.

According to a preferred specific embodiment of the invention, atransgenic pig may be produced by the methods as set forth in ExampleSection 6, infra. Briefly, estrus may be synchronized in sexually maturegilts (>7 months of age) by feeding an orally active progestogen (allyltrenbolone, AT: 15 mg/gilt/day) for 12 to 14 days. On the last day of ATfeeding all gilts may be given an intramuscular injection (IM) ofprostaglandin F_(2a) (Lutalyse: 10 mg/injection) at 0800 and 1600 hours.Twenty-four hours after the last day of AT consumption all donor giltsmay be administered a single IM injection of pregnant mare serumgonadotropin (PMSG: 1500 IU). Human chorionic gonadotropin (HCG: 750 IU)may be administered to all donors at 80 hours after PMSG.

Following AT withdrawal, donor and recipient gilts may be checked twicedaily for signs of estrus using a mature boar. Donors which exhibitedestrus within 36 hours following HCG administration may be bred at 12and 24 hours after the onset of estrus using artificial and natural(respectively) insemination.

Between 59 and 66 hours after the administration of HCG one- andtwo-cell ova may be surgically recovered from bred donors using thefollowing procedure. General anesthesia may be induced by administering0.5 mg of acepromazine/kg of bodyweight and 1.3 mg ketamine/kg ofbodyweight via a peripheral ear vein. Following anesthetization, thereproductive tract may be exteriorized following a mid-ventrallaparotomy. A drawn glass cannula (O.D. 5 mm, length 8 cm) may beinserted into the ostium of the oviduct and anchored to the infundibulumusing a single silk (2-0) suture. Ova may be flushed in retrogradefashion by inserting a 20 g needle into the lumen of the oviduct 2 cmanterior to the uterotubal junction. Sterile Dulbecco's phosphatebuffered saline (PBS) supplemented with 0.4% bovine serum albumin (BSA)may be infused into the oviduct and flushed toward the glass cannula.The medium may be collected into sterile 17×100 mm polystyrene tubes.Flushings may be transferred to 10×60 mm petri dishes and searched atlower power (50×) using a Wild M3 stereomicroscope. All one- andtwo-cell ova may be washed twice in Brinster's Modified Ova Culture-3medium (BMOC-3) supplemented with 1.5% BSA and transferred to 50 μldrops of BMOC-3 medium under oil. Ova may be stored at 38° C. under a90% N₂, 5% O₂, 5% CO₂ atmosphere until microinjection is performed.

One- and two-cell ova may be placed in a Eppendorf tube (15 ova pertube) containing 1 ml HEPES Medium supplemented with 1.5% BSA andcentrifuged for 6 minutes at 14000×g in order to visualize pronuclei inone-cell and nuclei in two-cell ova. Ova may then be transferred to a5-10 μl drop of HEPES medium under oil on a depression slide.Microinjection may be performed using a Laborlux microscope withNomarski optics and two Leitz micromanipulators. 10-1700 copies ofconstruct DNA (linearized at a concentration of about 1 ng/μl ofTris-EDTA buffer) may be injected into one pronuclei in one-cell ova orboth nuclei in two-cell ova.

Microinjected ova may be returned to microdrops of BMOC-3 medium underoil and maintained at 38° C. under a 90% N₂, 5% CO₂, 5% O₂ atmosphereprior to their transfer to suitable recipients. Ova may preferably betransferred within 10 hours of recovery.

Only recipients which exhibit estrus on the same day or 24 hours laterthan the donors may preferably be utilized for embryo transfer.Recipients may be anesthetized as described earlier. Followingexteriorization of one oviduct, at least 30 injected one-and/or two-cellova and 4-6 control ova may be transferred in the following manner. Thetubing from a 21 g×3/4 butterfly infusion set may be connected to a 1 ccsyringe. The ova and one to two mls of BMOC-3 medium may be aspiratedinto the tubing. The tubing may then be fed through the ostium of theoviduct until the tip reaches the lower third or isthmus of the oviduct.The ova may be subsequently expelled as the tubing is slowly withdrawn.

The exposed portion of the reproductive tract may be bathed in a sterile10% glycerol-0.9% saline solution and returned to the body cavity. Theconnective tissue encompassing the linea alba, the fat and the skin maybe sutured as three separate layers. An uninterrupted Halstead stitchmay be used to close the lina alba. The fat and skin may be closed usinga simple continuous and mattress stitch, respectively. A topicalantibacterial agent (e.g. Furazolidone) may then be administered to theincision area.

Recipients may be penned in groups of about four and fed 1.8 kg of astandard 16% crude protein corn-soybean pelleted ration. Beginning onday 18 (day 0=onset of estrus), all recipients may be checked daily forsigns of estrus using a mature boar. On day 35, pregnancy detection maybe performed using ultrasound. On day 107 of gestation recipients may betransferred to the farrowing suite. In order to ensure attendance atfarrowing time, farrowing may be induced by the administration ofprostaglandin F_(2a) (10 mg/injection) at 0800 and 1400 hours on day 112of gestation. In all cases, recipients may be expected to farrow within34 hours following PGF2a administration.

Twenty-four hours after birth, all piglets may be processed, i.e. earsnotched, needle teeth clipped, 1 cc of iron dextran administered, etc. Atail biopsy and blood may also be obtained from each pig.

Pigs produced according to this method are described in Example Section6, infra, and are depicted in FIG. 2. Such pigs are healthy, do notappear to be anemic, and appear to grow at a rate comparable to that oftheir non-transgenic littermates. Such pigs may transmit the transgeneto their offspring.

Pigs having certain characteristics may be especially useful for theproduction of human hemoglobin; such pigs, examples of which follow,represent preferred, non-limiting, specific embodiments of theinvention.

According to one preferred specific embodiment of the invention, atransgenic pig contains at least twenty copies of a globin transgene.

According to a second preferred specific embodiment, the P₅₀ of wholeblood of a transgenic pig according to the invention is increased by atleast ten percent over the P₅₀ of the whole blood of a comparablenon-transgenic pig,taking into consideration factors such as altitude,oxygen concentrations, pregnancy, the presence of mutant hemoglobin,etc. Thus, the present invention provides for a non-pregnant transgenicpig that carries and expresses a human globin transgene in which the P₅₀of whole blood of the transgenic pig is at least ten percent greaterthan the P₅₀ of whole blood of a comparable non-pregnant non-transgenicpig at the same altitude.

In other preferred specific embodiments, the present invention providesfor a transgenic pig in which the amount of human globin producedrelative to total hemoglobin is at least two percent, more preferably atleast five percent, and most preferably at least ten percent.

Section 6, infra, describes transgenic pigs which serve as workingexamples of preferred, non-limiting, specific examples of the invention.

Preparation of Human Hemoglobin and its Separation from Pig Hemoglobin

The present invention provides for a method for producing humanhemoglobin comprising introducing a transgene or transgenes encodinghuman hemoglobin, such as a human alpha globin and a human beta globingene, under the control of a suitable promoter or promoters, into thegenetic material of a pig so as to create a transgenic pig thatexpresses human hemoglobin in at least some of its blood cells.

The present invention also provides for a method of producing humanhemoglobin comprising (i) introducing a human alpha globin and a humanbeta globin gene, under the control of a suitable promoter or promoters,into the genetic material of a pig so as to create a transgenic pig thatexpresses human hemoglobin in at least some of its red blood cells; (ii)collecting red blood cells from the transgenic pig; (iii) releasing thecontents of the collected red blood cells to form a lysate; (iv)subjecting the lysate of the red blood cells to a purification procedurethat substantially separates human hemoglobin from pig hemoglobin; and(v) collecting the fractions that contain purified human hemoglobin.Such fractions may be identified by isoelectric focusing in parallelwith appropriate standards. In a preferred embodiment of the invention,human hemoglobin may be separated from pig hemoglobin by DEAE anionexchange column chromatography.

In order to prepare human hemoglobin from the transgenic pigs describedabove, red blood cells are obtained from the pig using any method knownin the art. The red blood cells are then lysed using any method,including hemolysis in a hypotonic solution such as distilled water, orusing techniques as described in 1981, Methods in Enzymology Vol. 76,and/or tangential flow filtration.

For purposes of ascertaining whether human hemoglobin is being producedby a particular transgenic pig, it may be useful to perform asmall-scale electrophoretic analysis of the hemolysate, such as, forexample, isoelectric focusing using standard techniques.

Alternatively, or for larger scale purification, human hemoglobin may beseparated from pig hemoglobin using ion exchange chromatography.Surprisingly, as discussed in Section 7, supra, human hemoglobin wasobserved to readily separate from pig hemoglobin using ion exchangechromatography whereas mouse hemoglobin and human hemoglobin were notseparable by such methods. Any ion exchange resin known in the art or tobe developed may be utilized, including, but not limited to, resinscomprising diethylaminoethyl, Q-Sepharose, QCPI (I.B.F.) Zephyr,Spherodex, ectiola, carboxymethylcellulose, etc. provided that the resinresults in a separation of human and pig hemoglobin comparable to thatachieved using DEAE resin.

According to a specific, nonlimiting embodiment of the invention, inorder to separate human from pig hemoglobin (including human/pighemoglobin hybrids) to produce substantially pure human hemoglobin, ahemolysate of transgenic pig red blood cells, prepared as above may beapplied to a DEAE anion exchange column equilibrated with 0.2 M glycinebuffer at pH 7.8 and washed with 0.2 M glycine pH 7.8/5 Mm NaCl, and maythen be eluted with a 5-30 Mm NaCl gradient, or its equivalent (see, forexample, Section 9 infra). Surprisingly, despite about 85 percenthomology between human and pig globin chains, human and pig hemoglobinseparates readily upon such treatment, with human hemoglobin elutingearlier than pig hemoglobin. Elution may be monitored by optical densityat 405 nm and/or electrophoresis of aliquots taken from serialfractions. Pig hemoglobin, as well as tetrameric hemoglobin composed ofheterodimers formed between pig and human globin chains, may beseparated from human hemoglobin by this method. Human hemoglobinproduced in a transgenic pig and separated from pig hemoglobin by thismethod has an oxygen binding capability similar to that of native humanhemoglobin.

According to another specific, non-limiting embodiment of the invention,human hemoglobin may be separated from pig hemoglobin (includinghuman/pig hemoglobin hybrids) using QCPI ion exchange resin as follows:

About 10 mg of hemoglobin prepared from transgenic pig erythrocytes maybe diluted in 20 ml of Buffer A (Buffer A=10 mM Tris, 20 mM Glycine pH7.5). This 20 ml sample may then be loaded at a flow rate of about 5ml/min onto a QCPI column (10 ml) which has been equilibrated withBuffer A. The column may then be washed with 2 volumes of Buffer A, andthen with 20 column volumes of a 0-50 mM NaCl gradient (10 columnvolumes of Buffer A+10 column volumes of 10 mM Tris, 20 mM Glycine, 50mMNaCl pH 7.5) or, alteratively, 6 column volumes of 10 mM Tris, 20 mMGlycine, 15 mM NaCl, pH 7.5, and the O.D.₂₈₀ absorbing material may becollected in fractions to yield the separated hemoglobin, humanhemoglobin being identified, for example, by isoelectric focusing usingappropriate standards. The QCPI column may be cleaned by elution with 2column volumes of 10 mM Tris, 20 mM Glycine, 1M NaCl, pH 7.5.

For certain mutant hemoglobins, it may be desirable to utilize amodified purification procedure. Accordingly, for the separation of HbPresbyterian from pig Hb, a procedure as described in Example Section12.1, infra, may be used, and for separation of Hb Yoshizuka, aprocedure as described in Example Section 12.2, infra, may be used.

Preparation of Human/Pig Hybrid Hemoglobin

The present invention also provides for essentially purified andisolated human/pig hybrid hemoglobin, in particular human α/pig β hybridhemoglobin. Pig α/human β hybrid has not been observed to form either invitro in reassociation experiments or in vivo in transgenic pigs.

The present invention provides for hybrid hemoglobin and its use as ablood substitute, and for a pharmaceutical composition comprising theessentially purified and isolated human/pig hemoglobin hybrid in asuitable pharmacological carrier.

Hybrid hemoglobin may be prepared from transgenic pigs, as describedherein, and then purified by chromatography, immunoprecipitation, or anyother method known to the skilled artisan. The use of isoelectricfocusing to separate out hemoglobin hybrid is shown in FIGS. 3 and 5.

Alternatively, hybrid hemoglobin may be prepared using nucleic acidconstructs that comprise both human and pig globin sequences which maythen be expressed in any suitable microorganism, cell, or transgenicanimal. For example, a nucleic acid construct that comprises the human αand pig β globin genes under the control of a suitable promoter may beexpressed to result in hybrid hemoglobin. As a specific example, human αglobin and pig β globin genes, under the control of cytomegaloviruspromoter, may be transfected into a mammalian cell such as a COS cell,and hybrid hemoglobin may be harvested from such cells. Alternatively,such constructs may be expressed in yeast or bacteria.

It may be desirable to modify the hemoglobin hybrid so as to render itnon-immunogenic, for example, by linkage with polyethylene glycol or byencapsulating the hemoglobin in a membrane, e.g. in a liposome.

EXAMPLE Generation of Transgenic Pigs that Produce Human HemoglobinMaterials and Methods Nucleic Acid Constructs

Constructs 116 (the ααβ construct), 185 (the αpβ construct), 263 (theαpδ construct) 339, 293 and 294 were microinjected into pig ova as setforth below in order to produce transgenic pigs.

Production of Transgenic Pigs

Estrus was synchronized in sexually mature gilts (>7 months of age) byfeeding an orally active progestogen (allyl trenbolone, AT: 15mg/gilt/day) for 12 to 14 days. On the last day of AT feeding all giltsreceived an intramuscular injection (IM) of prostaglandin F_(2a)(Lutalyse: 10 mg/injection) at 0800 and 1600. Twenty-four hours afterthe last day of AT consumption all donor gilts received a single IMinjection of pregnant mare serum gonadotropin (PMSG: 1500 IU). Humanchorionic gonadotropin (HCG: 750 IU) was administered to all donors at80 hours after PMSG.

Following AT withdrawal, donor and recipient gilts were checked twicedaily for signs of estrus using a mature boar. Donors which exhibitedestrus within 36 hours following HCG administration were bred at 12 and24 hours after the onset of estrus using artificial and natural(respectively) insemination.

Between 59 and 66 hours after the administration of HCG, one- andtwo-cell ova were surgically recovered from bred donors using thefollowing procedure. General anesthesia was induced by administering 0.5mg of acepromazine/kg of bodyweight and 1.3 mg ketamine/kg of bodyweightvia a peripheral ear vein. Following anesthetization, the reproductivetract was exteriorized following a mid-ventral laparotomy. A drawn glasscannula (O.D. 5 mm, length 8 cm) was inserted into the ostium of theoviduct and anchored to the infundibulum using a single silk (2-0)suture. Ova were flushed in retrograde fashion by inserting a 20 gneedle into the lumen of the oviduct 2 cm anterior to the uterotubaljunction. Sterile Dulbecco's phosphate buffered saline (PBS)supplemented with 0.4% bovine serum albumin (BSA) was infused into theoviduct and flushed toward the glass cannula. The medium was collectedinto sterile 17×100 mm polystyrene tubes. Flushings were transferred to10×60 mm petri dishes and searched at lower power (50×) using a Wild M3stereomicroscope. All one- and two-cell ova were washed twice inBrinster's Modified Ova Culture-3 medium (BMOC-3) supplemented with 1.5%BSA and transferred to 50 μl drops of BMOC-3 medium under oil. Ova werestored at 38° C. under a 90% N₂, 5% O₂, 5% CO₂ atmosphere untilmicroinjection was performed.

One- and two-cell ova were placed in an Eppendorf tube (15 ova per tube)containing 1 ml HEPES Medium supplemented with 1.5% BSA and centrifugedfor 6 minutes at 14000×g in order to visualize pronuclei in one-cell andnuclei in two-cell ova. Ova were then transferred to a 5-10 μl drop ofHEPES medium under oil on a depression slide. Microinjection wasperformed using a Laborlux microscope with Nomarski optics and two Leitzmicromanipulators. 10-1700 copies of construct DNA (1 ng/μl of Tris-EDTAbuffer) were injected into one pronuclei in one-cell ova or both nucleiin two-cell ova.

Microinjected ova were returned to microdrops of BMOC-3 medium under oiland maintained at 38° C. under a 90% N₂, 5% CO₂, 5% O₂ atmosphere priorto their transfer to suitable recipients. Ova were transferred within 10hours of recovery.

Only recipients which exhibited estrus on the same day or 24 hours laterthan the donors were utilized for embryo transfer. Recipients wereanesthetized as described earlier. Following exteriorization of oneoviduct, at least 30 injected one- and/or two-cell ova and 4-6 controlova were transferred in the following manner. The tubing from a 21 g×3/4butterfly infusion set was connected to a 1 cc syringe. The ova and oneto two mls of BMOC-3 medium were aspirated into the tubing. The tubingwas then fed through the ostium of the oviduct until the tip reached thelower third or isthmus of the oviduct. The ova were subsequentlyexpelled as the tubing was slowly withdrawn.

The exposed portion of the reproductive tract was bathed in a sterile10% glycerol-0.9% saline solution and returned to the body cavity. Theconnective tissue encompassing the linea alba, the fat and the skin weresutured as three separate layers. An uninterrupted Halstead stitch wasused to close the lina alba. The fat and skin were closed using a simplecontinuous and mattress stitch, respectively. A topical antibacterialagent (Furazolidone) was then administered to the incision area.

Recipients were penned in groups of four and fed 1.8 kg of a standard16% crude protein corn-soybean pelleted ration. Beginning on day 18 (day0=onset of estrus), all recipients were checked daily for signs ofestrus using a mature boar. On day 35, pregnancy detection was performedusing ultrasound. On day 107 of gestation recipients were transferred tothe farrowing suite. In order to ensure attendance at farrowing time,farrowing was induced by the administration of prostaglandin F_(2a) (10mg/injection) at 0800 and 1400 hours on day 112 of gestation. In allcases, recipients farrowed within 34 hours following PGF2aadministration.

Twenty-four hours after birth, all piglets were processed, i.e. earswere notched, needle teeth clipped, 1 cc of iron dextran wasadministered, etc. A tail biopsy and blood were also obtained from eachpig.

Results and Discussion

Of 3566 injected ova, thirteen transgenic pigs that expressed humanhemoglobin were born, two of which died shortly after birth due tonormal breeding-related incidents completely unrelated to the fact thatthey were transgenic pigs (Table I). The remaining 11 appeared to behealthy. A photograph of one transgenic pig is presented in FIG. 2.Profiles of the pigs and of the percent "authentic" and "hybrid" humanhemoglobin ("HB") produced are set forth in Table II, infra. Totalhemoglobin was calculated as the sum of human αβ plus one-half of thehuman α pig β hybrid. FIGS. 3A and 3B presents the results ofisoelectric focussing and triton acid urea gels of hemoglobin producedby three of these pigs (numbers 12-1, 9-3, and 6-3) which demonstratethe expression of human alpha and beta globin in these animals.

                  TABLE I                                                         ______________________________________                                        Efficiency of Transgenic Pig Production                                         Human Hemoglobin Gene Construct(s)                                                                     Total After                                          Parameter 22 Trials                                                         ______________________________________                                        Total Ova Collected    8276                                                     Total # Fertilized 7156                                                       Total # Injected 3566                                                         # Injected Ova Transferred 3566                                               # Control Ova Transferred 279                                                 # Recipients Used 104                                                         # Pigs Born (Male, Female) 208, 332                                           # Transgenic (Male, Female) 8, 5 (0.36).sup.a                                 # Expressing 13                                                             ______________________________________                                         .sup.a Proportion of injected ova which developed into transgenic pigs (1     transgenics/3566 injected ova).                                          

                  TABLE II                                                        ______________________________________                                        FOUNDERS                                                                                               AUTH-                                                     ENTIC HY- TOTAL                                                             GEN- TRANSGENE HUMAN BRID HUMAN COPY                                         PIG DER CONSTRUCT HB HB HB #                                                ______________________________________                                        6-3  F      116        6.2%   8.1%  10.3%  57                                   9-3 F 116 1.0% 33.1% 16.6% 1                                                  22-2 M 185 <1% 5.0% 5.0% 55                                                 33-7 F      185        *died shortly after birth                                                                     0.5                                    38-1 F      185        1.0.%  8.3%  5.2%   17                                   38-3 M 185 4.7% 17.2% 13.2% 22                                                38-4 M 185 3.2% 7.0% 6.7% 5                                                   47-3 M 263 <1% 2.9% 2.0% 4-6                                                  47-4 F 263 <1% 18.5% 10.0% 1-2                                                52-3 M 263 <1% 7.6% 4.0%                                                      52-7 M 263 <1% 26.4% 13.0%                                                    53-11 M 263 <1% 15.5% 8.0%                                                    70-3 F 339 23 31 38 3                                                       ______________________________________                                    

Table III presents the profiles of offspring of pig number 9-3, whichshows that the F1 generation of transgenic pigs are capable ofexpressing hemoglobin. Of note, none of the offspring of pig number 6-3were found to be transgenic, possibly due to the absence of transgene inthe animal's reproductive tissue.

Table IV presents hemoglobin expression data of offspring of pig 38-4carrying the "185" construct (the "αpβ" construct; see FIG. 1B). Table Vpresents a summary of the profiles of offspring of pig number 38-4 inwhich a large percentage (37.1%) of offspring were positive forexpression of human hemoglobin indicating germ line transmission of thetransgene. FIG. 19 presents the results of isoelectric focussing whichdemonstrates the levels of hemoglobin expression in representativetransgene positive 38-4 offspring.

                  TABLE III                                                       ______________________________________                                        F1 (OFFSPRING) OF PIG 9-3                                                                             AUTH-   HY-                                                ENTIC BRID TO-                                                              GEN-  HUMAN HUMAN TAL COPY                                                   PIG DER CONST. HB HB HUM. #                                                 ______________________________________                                        9-3-1 F      116      1.0%    31.5%  16.0% 1                                    9-3-2* F 116 1.0% 32.9% 17.0% 1                                               9-3-3 M 116 1.0% 29.7% 15.0% 1                                                9-3-4 M 116 1.0% 32.8% 17.0% 1                                                9-3-6 F 116 1.0% 29.1% 15.0% 1                                                9-3-8 M 116 1.0% 31.6% 16.0% 1                                                9-3-9 M 116 1.0% 30.2% 16.0% 1                                              ______________________________________                                         *9-3-2 died the day after birth.                                         

                  TABLE IV                                                        ______________________________________                                        EXPRESSION DATA PER LITTER FOR TRANSGENIC PIGS                                  CARRYING THE "185" CONSTRUCT                                                          Litter            %                                                   Founder No. Gilt Pigs Positive #Tq Avg. Authentic HbA                       ______________________________________                                        38-4  1      544     10   20.0% 2    8.8%                                        2 213 11 45.4% 5                                                              3 882 5 20.0% 1 10.9%                                                         4 4923 6 83.3% 5 9.4%                                                         5 710 6 75.0% 4 4.5%                                                          6 978 11 36.4% 4 7.1%                                                         7 466 4 25.0% 1 3.6%                                                          8 464 15 33.3% 5 5.1%                                                         9 461 8 62.5% 5 6.6%                                                          10 1657 10 30.0% 3 9.0%                                                       11 892 3 33.3% 1 5.7%                                                         12 995 11 27.3% 3 4.4%                                                        13 209 11 36.4% 4 5.4%                                                        14 424 10 30.0% 3 5.9%                                                        15 1659 14 35.7% 5 4.4%                                                       16 420 12 8.3% 1 2.0%                                                         17 373 7 28.6% 2 11.8%                                                        18 497 8 62.5% 5 6.0%                                                         19 742 8 25.0% 2 1.0%                                                         20 1420 14 42.9% 6 8.1%                                                       21 41 5 40.0% 2 1.0%                                                          22 540 11 36.4% 4 5.3%                                                        23 7114 11 54.5% 6 3.4%                                                       24 744 11 27.3% 3 4.9%                                                        25 600 14 42.9% 6 5.5%                                                        26 1180 9 44.4% 4 2.0%                                                        27 1137 12 25.0% 3 6.1%                                                       28 970 8 37.5% 3 10.8%                                                        29 78 6 0 0                                                                   30 214 14 50.0% 7 5.5%                                                        31 279 6 50.0% 3 10.3%                                                        32 281 11 45.5% 5 5.1%                                                           33     21-474  6    33.3% 2    12.3%                                       34 1151 10 30.0% 3 5.3%                                                         318  118                                                                 ______________________________________                                    

                  TABLE V                                                         ______________________________________                                        38-4 BREEDING SUMMARY                                                         ______________________________________                                        FOUNDER   LITTERS    PIGLETS    PIGS/LITTER                                   ______________________________________                                          38-4(M) 34 318 9.4                                                          TRANSGENIC                                                                              FREQUENCY  AVG. AUTHENTIC HbA                                                                            MALES                                    ______________________________________                                          118 37.1% 6.2% 59                                                           AUTHENTIC HUMAN HB                                                                           FE-      AUTHENTIC HUMAN HB                                      EXPRESSION LEVEL MALES EXPRESSION LEVEL                                     ______________________________________                                          5.7% 59 6.8%                                                                ______________________________________                                    

The birth weights of the transgenic pigs have been approximatelyequivalent to the birth weights of their non-transgenic littermates. Asthe transgenic pigs matured, their weights remained comparable to theweights of control animals.

EXAMPLE Separation of Human Hemoglobin from Pig Hemoglobin by DeaeChromatography Materials and Methods Purification by Deae Chromatography

For purification, red blood cells were collected by centrifugation of5000 rpm for 3 minutes in an eppendorf microcentrifuge and washed threetimes with an equal volume (original blood) of 0.9% NaCl. Red cells werelysed with 1.5 volumes deionized H₂ O, centrifuged at 15,000 rpm, andthe supernatant was fractionated by anion exchange chromatography. DEAEcellulose chromatography (DE-SE manufactured by Whatman, Ltd.) wasperformed according to W. A. Schroeder and T. H. J. Huisman "TheChromatography of Hemoglobin", Dekker, N.Y., pp. 74-77. The 0.25 ml redcell hemolysate described above was applied to 1 cm×7 cm DE-52 columnpre-equilibrated in 0.2 M glycine Ph 7.8 and was washed with 5 columnvolumes of 0.2 M glycine Ph 7.8/5 Mm NaCl. Hemoglobins were eluted witha 200 ml 5-30 mM NaCl/0.2 M glycine pH 7.8 gradient. To complete elutionof pig hemoglobin, an additional 50 to 100 ml of 30 mM CaCl/glycine pH7.8 was added to the column. Elution of hemoglobin was monitored byabsorbance of 415 mM and by IEF analysis of column fractions.

Reassociation of Globin Chains

Reassociation of globin chains was performed essentially as described inMethods in Enzymol. 76:126-133. 25 lambda of pig blood, 25 lambda ofhuman blood, or a 25 lambda mixture of 12.5 lambda human blood and 12.5lambda pig blood were treated as follows. The blood was pelleted at asetting of 5 on microfuge for 2 minutes, then washed three times with100 lambda 0.9 percent NaCl. The cells were lysed with 50 lambda H₂ O,then spun at high speed to confirm lysis. 50 lambda of the lysed cellswas then combined with 50 lambda 0.2 M Na Acetate, pH 4.5, put on iceand then incubated in a cold room overnight. After adding 1.9 ml 0.1 MNaH₂ PO₄ 4, pH 7.4 each sample was spun in centricon tubes at 4° C. and5 K until about 0.5 ml remained. Then 1 ml of 0.1 M NaH₂ PO₄ pH 7.4 wasadded and spun through at about 5 K until about 0.2 ml volume was left.The hemoglobin was then washed from the walls of the centricon tube, aneppendorf adaptor was attached, and a table top microfuge was used toremove each sample from its centricon tube. The samples were thenanalyzed by isoelectric focusing.

Results and Discussion Human and Pig Hemoglobin were Separated from aHemolyzed Mixture of Human and Pig Blood

Equal proportions of human and of pig blood were mixed and lysed, andthe resulting hemolysate was subjected to DEAE chromatography asdescribed supra. As shown in FIG. 4A, pig hemoglobin separated virtuallycompletely from human hemoglobin. This complete separation is surprisingin light of the structural similarity between human and pig hemoglobin;pig and human alpha globin chains are 84.4 percent homologous and pigand human beta globin chains are 84.9 percent homologous. It is furthersurprising because, as shown in FIG. 4C, when human and mouse blood wasmixed, hemolyzed, applied to and eluted from a DEAE column according tomethods set forth in Section 7.1.1., supra, human and mouse hemoglobinwere not observed to separate despite the fact that mouse and humanalpha globin chains are about 85.8 percent homologous and mouse andhuman beta globin chains are 80.1 percent homologous. The ease ofseparation of human and pig hemoglobin on DEAE resin appears to be bothefficient and economical.

Interestingly, the order of elution of the proteins from the anionexchange column was not as expected. Based on the relative pI's of theproteins as deduced from the IEF gels, the predicted order of elutionwould be first the hybrid (human α/pig β) followed by the authentichuman α/human β. The last protein to elute from the anion exchangecolumn then would be the endogenous pig α/pig β protein. However, underall the conditions currently attempted the order of elution was alteredsuch that the human hemoglobin was the first to elute. The second peakwas an enriched fraction of the hybrid followed very closely by the pighemoglobin.

Human and Pig Hemoglobin and Human/Pig Heterologous Hemoglobin wereSeparated from Hemolysate Prepared from a Transgenic Pig

Blood from transgenic pig 6-3 (as described in Section 6, supra) waslysed by hypotonic swelling and the resulting hemolysate was subjectedto DEAE chromatography as described supra. As shown in FIG. 4B, humanhemoglobin was separated from pig hemoglobin and from human a globin/pigbeta globin heterologous hemoglobin. As shown in FIG. 4D, humanhemoglobin was substantially purified by this method.

Pig Alpha Globin/Human Beta Globin Heterologous Hemoglobin does notAppear to Form Based on Reassociation Data

Heterologous association between pig alpha globin and human beta globinchains has not been detected in hemolysates obtained from humanhemoglobin-expressing transgenic pigs. It was possible, however, thatthis observation could be explained by relatively low levels of humanbeta globin expression. Alternatively, association between pig alphaglobin and human beta globin may be chemically unfavorable. In order toexplore this possibility, reassociation experiments were performed inwhich pig and human hemoglobin were mixed, dissociated, and then theglobin chains were allowed to reassociate. As shown in the isoelectricfocusing gels depicted in FIG. 5, although pig α/pig β, human α/human β,and human α/pig β association was observed, no association between pig αglobin and human β globin appeared to have occurred. Therefore the pigα/human β heterologous hemoglobin should not be expected to complicatethe purification of human hemoglobin from transgenic pigs.

EXAMPLE Separation of Human Hemoglobin from Pig Hemoglobin by QCPIChromatography Materials and Methods

Clarified hemolysate from transgenic pig 6-3 13 mg/ml; Buffer A: 10 mMTris, 20 mM Glycine pH 7.5; Buffer B: 10 mM Tris, 20 mM Glycine, 15 mMNaCl pH 7.5; Buffer C: 10 mM Tris, 20 mM Glycine, 1M NaCl pH 7.5; BufferD: 10 mM Tris, 20 mM Glycine, 50 mM NaCl pH 7.5; QCPI column 10 mlEquilibrated in Buffer A; Trio purification system. 10 mg of hemoglobinprepared from transgenic pig 6-3 was diluted in 20 ml Buffer A. 20 ml ofsample was loaded at a flow rate of 5 ml/min onto the QCPI column, andwashed with 2 column volumes of Buffer A. The column was then washedwith 20 column volumes of a 0-50 mM NaCl gradient. (10 column volumesBuffer A+10 column volumes of Buffer D) and the O.D.₂₈₀ absorbingmaterial was collected. The column was then cleaned with 2 columnvolumes of Buffer C, and then re-equilibrated with 2 column volumes ofBuffer A.

Results

Analysis of the UV trace (peak vs. volume of gradient) (FIG. 6) revealedthat the human hemoglobin was eluted at 15 mM NaCl. Subsequentpurifications have been performed utilizing the same protocol as above,only using 6 column volumes of Buffer B (15 mM NaCl) to elute the humanhemoglobin rather than the gradient. In addition, non-transgenic pigchromatographed by this method does not elute from the QCPI with BufferB, while native human hemoglobin does. The protein that eluted at 15 mMNaCl was analyzed on the Resolve isoelectric focussing system and foundto be essentially pure of contaminating pig hemoglobin or hybridhemoglobin.

EXAMPLE Human Alpha/Pig Beta Globin Hybrid Hemoglobin Exhibit IncreasedP₅₀

As shown in Tables II and III, supra, transgenic pigs of the inventionwere all found to produce significant amounts of human α/pig β globinhybrid hemoglobin (the pig α/human β hybrid was not observed).Significantly, pigs that expressed higher percentages of hybrid alsoappeared to exhibit elevated P₅₀ values for their whole blood (FIG. 7).

EXAMPLE Enhanced Expression Using Pig Beta Globin Regulatory Sequences

The 339 construct (FIGS. 1R and 12) containing the pig adult beta globingene promoter region (FIG. 8), was used to prepare transgenic pigsaccording to the method set forth in Section 6.1.2. supra. FIG. 15depicts an isoelectric focusing gel analysis of hemoglobin produced bypig 70-3; equal amounts of hemoglobin from transgenic pig 6-3, carryingthe 116 construct (FIG. 1A) and human hemoglobin are run in adjacentlanes for comparison. As indicated by the brighter bands observed in thelane containing pig 70-3 hemoglobin at positions corresponding to humanand hybrid hemoglobins (relative to the lane containing pig 6-3hemoglobin), the amount of human hemoglobin produced by pig 70-3 isgreater than the amount produced by pig 6-3. It has been calculated that38 percent of the total hemoglobin produced by pig 70-3 is humanhemoglobin, whereas 10 percent of total hemoglobin produced by pig 6-3is human hemoglobin (see Table II and Section 6.2. supra, for data andcalculations). This suggests that the pig beta globin promoter region ismore efficient than the human beta globin promoter in transgenic pigs.

In a separate series of experiments, two more transgenic pigs,expressing human hemoglobin, were obtained using construct "339" (pigs80-4 and 81-3) (FIG. 17). Human hemoglobin levels in these transgenicpigs was determined by running isoelectric focussing gels anddensitometric scanning of the individual bands (FIG. 18). As indicatedin FIG. 17, both pig 70-3 and pig 80-4 expressed high levels ofauthentic human hemoglobin. To obtain the copy number of transgenes,genomic DNA (isolated from the tail) was digested with EcoR I and aSouthern Blot was performed. The probe used was a 427 bp NcoI/Bam HIfragment of human beta globin gene containing the first exon, firstintron and part of the second exon.

EXAMPLE Molecular Modeling of Pig Hemoglobin and the α₁ β₁ Interface ofa Hybrid Between Pig β Human α Globin

It has been found that the amount of hybrid human α/pig β hemoglobinoften exceeds the amount of human hemoglobin. The molecular basis ofthis observation has been investigated using molecular modeling andmolecular biology. The model structure of the hybrid molecule is basedon the known structures of human hemoglobins and the structural homologybetween the human and pig structures (A. M. Lesk, 1991, ProteinArchitecture: A Practical Approach, Oxford University Press, N.Y.). Thepig and hybrid hemoglobin structures were modeled using the followingfour steps: (1) hydrogen atoms were added to the X-ray model and theirpositions modified using energy minimization; (2) amino acid residuereplacements were introduced to model the target pig and hybridstructures (no chain alignment was necessary); (3) the side chainpositions of these modified residues were energy minimized; and (4) theresult was visually examined and found to be sound. The modeledstructures are shown in FIG. 20.

Detailed examination of all the relevant contacts indicated strikingdifferences at several residues. For example, at position β112 the humanhemoglobin has a cysteine residue but the hybrid has a valine residue.The valine is in apparent closer contact (arrow in FIG. 20) with theopposing subunit, and thus may be more effective in stabilizing the α₁β₁ interface (FIG. 21).

The effect of amino acid substitutions at the α₁ β₁ interface on thehydrophobic and polar interactions as predicted by HINT are shown inTABLE VI. HINT is software from Virginia Commonwealth UniversityLicensed from Medical College of Virginia, Richmond, Va. that cananalyze the positive and negative scores as determined by attractive andrepulsive interactions known from experimental physical chemistrymeasurements. TABLE VI represents the differences between the unmodifieddimer and the one with the specified replacement. TABLE VII has the sameformat as TABLE VI with the following two exceptions: (1) as eachreplacement is added, the previous one(s) are kept, and (2) the reporteddifference is a comparison between the current dimer and the onereflected in the preceding row. As the subsequential changes are made,the predicted attractive forces at the interface increase. If eachcolumn is summed up the total difference between the unmodified dimerand the one with seven changes is obtained. The sums are +1340 forhydrophobic and +660 for polar.

                  TABLE VI                                                        ______________________________________                                        Effect of amino acid replacements at the α1β1                        interface                                                                                                  Predicted Difference                           Chain  Residue    Replacement                                                                              Hydrophobic                                                                           Polar                                    ______________________________________                                        α                                                                              30         E to T     +250    +10                                        α 36 F to Y -110 +220                                                   α 106 L to F +20 +10                                                    α 107 V to S -10 +120                                                   α 107 V to C 0 +150                                                     α 111 A to C +30 +100                                                   β 33 V to L +70 0                                                        β 112 C to V +330 -60                                                    β 112 C to I +360 -50                                                    β 115 A to V +80 +10                                                     β 115 A to L +90 +10                                                     β 119 G to H +250 +120                                                   β 125 P to M 80 0                                                        β 128 A to I +80 0                                                       β 131 Q to E +120 +110                                                 ______________________________________                                    

                  TABLE VII                                                       ______________________________________                                        Effect of combinations of amino acid replacements                               at the α1β1 interface on the hydrophobic and polar interactio    ns                                                                                                           Predicted Difference                           Chain  Residue    Replacement                                                                              Hydrophobic                                                                           Polar                                    ______________________________________                                        β 112        C to I     +360    -50                                        α 110 A to I +200 +10                                                   β 115 A to V +150 +10                                                    β 119 G to H +270 +130                                                   α 36 F to Y -130 +240                                                   β 33 V to L +80 +0                                                       α 30 E to T +260 +10                                                    β 131 Q to E +150 +310                                                 ______________________________________                                    

EXAMPLE Expression of Genetically Modified Hemoglobins in TransgenicAnimals

Of the known human hemoglobin variants, about two dozen exhibit a loweroxygen affinity, which could be advantageous in clinical applications.While many of these mutants result in unstable hemoglobin molecules,several variants have desirable biochemical properties and can be usedfor the generation of blood substitutes using recombinant DNAtechnology. Transgenic pigs expressing two of these variants, HbPresbyterian (108 Asn→Lys, FIG. 1G) and Hb Yoshizuka (108 Asn→Asp, FIG.1F) have been produced and purification and characterization of theexpressed human globins is described below.

Purification and Characterization of Hb Presbyterian

The amino acid substitution generated in Hb Presbyterian (β108 Asn→Lys)results in the comigration of Hb Presbyterian with the hybrid (hαpβ)hemoglobin on isoelectric focussing gels. Based on previous results withthe purification of human hemoglobin from hybrid and porcine hemoglobinsand the more positive nature of the Hb Presbyterian it should be easierto purify this variant hemoglobin on an anion exchange resin.Approximately 500 ml of blood was obtained from the transgenic pig57-10. The blood was washed several times with isotonic saline and thenlysed by hypotonic swelling in water. The cell membranes were removed bycentrifugation at 10000 ×g to yield a final hemoglobin concentration ofabout 100 mg/ml. Hb Presbyterian was purified from the hybrid andporcine hemoglobins as follows: 1-2.5 g of hemolysate was loaded onto anXK 50/30 column packed with 450 ml of Biorad Macroprep High Q resinequilibrated with 10 mM Tris-Cl and 20 mM Glycine at pH 8.1 (Buffer A).The proteins were eluted at a flow rate of 10 ml/min with a linear saltgradient of 9-16% Buffer B (Buffer A containing 250 mM NaCl) over 3000ml.

The initial peak was thought to be Hb Presbyterian followed by theco-elution of the hybrid and porcine hemoglobins (FIG. 20). To confirmthe identity of the first peak as Hb Presbyterian and not the hybridhemoglobin, a sample of the protein was run on Reversed Phase HPLC (FIG.21). The initial peak from the anion exchange column was Hb Presbyterianwith the α-chains eluting at the same time as normal human α-chains andthe β-chains eluting slightly faster than normal human β-chains. Thiswas also found to be an excellent way of determining if porcinehemoglobin was contaminating the column fractions. Using thispurification procedure and the analysis on HPLC the recombinant HbPresbyterian derived from the transgenic pig 58-10 was judged to begreater than 95% pure.

Purified Hb Presbyterian was dialyzed against 50 mM HEPES and 100 mMNaCl at pH 7.4 and oxygen equilibrium curves determined using a HemoxAnalyzer (TCS Products, Southampton, Pa.). The Hemox Analyzer wasmodified to allow analog to digital data conversion for ease of oxygenbinding calculations. Under these conditions the Hb Presbyterian had aP₅₀ of 25.8 mmHg (Hill Coefficient n=2.3) versus 13.3 mm Hg (n=2.9) forHb A indicating that the Hb Presbyterian bound oxygen with loweraffinity than native Hb. Preliminary results to determine the BohrEffect (Influence of pH on the oxygen affinity) indicated a normal Bohreffect for Hb Presbyterian (FIG. 22).

Purification and Characterization of Hb Yoshizuka

Blood samples taken from the transgenic pigs expressing Hb Yoshizuka(68-3 and 68-2) were treated essentially the same as described above.The final concentration of the hemolysate was approximately 100 mg/ml.The purification of the protein required a slightly different strategy,however. A sample of hemolysate from 68-3 (about 10 mg) was loaded ontoan HR 10/30 Biorad Macroprep High Q resin column equilibrated with 10 mMTris-Cl and 20 mM Glycine at pH 8.7 (Buffer A). The hemoglobins wereeluted at 2.5 mls/min with a 5-30% linear gradient of Buffer B (Buffer Aplus 250 mM NaCl) over 500 ml (FIG. 23). Fractions were collected andanalyzed by IEF to assess purity which was determined to be about 75% orbetter.

EXAMPLE Cloning of Porcine β Globin Locus Control Regions (LCR)

The porcine β Locus Control Region (LCR) was cloned and sequenced.Constructs comprising the human globin genes under the control of theporcine LCR may be used to generate transgenic pigs with enhancedhemoglobin expression.

A porcine genomic library in EMBL-3 (Clonetech, Calif.) was plated and 2million plaques were screened. A 3 kb Sal I to Eco RI fragment(extending from -1.9 kb to -4.9 kb with respect to porcine ξ gene)derived from the 12 Kb SALI fragment of ξ gene was used as a probe. Twopositive clones (Phage L and Phage H) were isolated.

Southern analysis of restricted Phage L and Phage H suggested that thetwo clones overlapped (FIG. 26A). The 7 kb and 4 kb SSt1 fragments ofPhage H were subcloned into plasmid pGem3 to obtain plasmids pPH2 andpPH1, respectively (deposited with the ATCC and assigned accessionnumbers 75519 for pPH2 and 75519 for pPH1). These plasmids weresequenced (from Sp6 and T7 promoter) and the sequence was compared withthe human genomic sequences. All the matches were with the sequence ofthe human beta globin region located on chromosome 11, which containsthe entire beta globin locus. Further sequencing was carried out for PH1using additional primers. Sequence analysis revealed that the 3' end ofclone PH1 (PH1-TA1, FIG. 27A) was 69% homologous to human LCRI (FIG.27B). The sequence of the 5' end of PH1 and 3' end of PH2 were joined(joined plcr2, FIG. 28) and found to be similar to human LCRII (FIG.29). The 5' end of PH2 (PH2-T7, FIG. 30A) had a stretch of 38 bp whichwas 78.9% homologous to a sequence in human LCRIV (FIG. 30B).

EXAMPLE Optimization of Human β Globin Gene

Analysis of blood samples from transgenic pigs carrying human hemoglobingenes indicates that human α-globin is expressed at higher levels thanhuman beta globin. The overall production of human hemoglobin tetramersin transgenic animals may be increased by optimizing the expression ofhuman β-globin gene expression. Such optimization may improve expressionof β-globin by affecting mRNA structure, stability or rate oftranslation.

One approach to increasing the level of expressed β-globin is toengineer the human β-globin gene, from the promoter region through thecoding sequence and into the polyadenylation site and 3' untranslatedregion, to be similar to the pig β-globin gene, but without altering theamino acid sequence from that of the authentic wild-type human β-globin.

Using polymerase chain reactions, synthetic oligonucleotides andrestriction digests, constructs were genetically engineered to optimizethe human β-globin gene for porcine expression. As shown in FIG. 31, thepromoter region, intervening sequences I and II (IVSI and IVSII), aswell as poly A and 3' UTR region are pig sequences and were obtained byrestriction digests from pig β-globin gene. Exon 1, Exon 2 and Exon 3were generated either by polymerase chain reaction or by oligonucleotidesynthesis (exon 2 SfaN1 through Bam HI, and all of exon 3).

A comparison of coding sequences of optimized, human and pig sequencesis diagrammed in FIG. 32. Lines in the optimized sequence indicatesnucleic acid changes from the human sequence.

Table VIII shows the number of changes between human, optimized and pigcoding sequences. The Table is subdivided into the 3 Exons and showschanges at the nucleotide, codon and amino acid level.

                  TABLE VIII                                                      ______________________________________                                        Optimization of β Globin Codons                                                       Number of changes                                                         Exon 1      Exon 2      Exon 3                                                H→H*                                                                           H→P                                                                            H→H*                                                                         H→P                                                                          H→H*                                                                         H→P                         ______________________________________                                        Nucleotides                                                                            5       14      18    31    8     24                                   Codons 5 10 18 25 8 18                                                        Amino acids 0 5 0 7 0 10                                                    ______________________________________                                         H* = Optimized human β globin gene                                  

Comparisons between the human and pig β-globin coding sequences aredepicted in FIG. 35. Differences are signified by small letters in thepig (bottom) sequence and codons containing nucleotide changes areunderlined. Comparisons of human and optimized β-globin coding sequencesand optimized and pig β-globin coding sequences are shown in FIGS. 36and 37, respectively. The coding sequence and amino acid sequence ofoptimized β-globin gene are indicated in FIG. 38. A plasmid containingthe optimized β-globin gene, designated pGEM3B*Δ3', has been depositedwith the ATCC and assigned accession number 75518.

A number of constructs were engineered to express the optimized β-globingene. Construct 505 (FIG. 33) contains the human locus control region,the human α-globin gene driven by its own promoter, the human ξ-globingene also driven by its own promoter, and the optimized β-globin genewhich has the optimized coding region. The gene order in this constructis LCRαξβ (where * signifies optimized β gene). A second construct,designated Construct 515 (FIG. 34), contains the human locus controlregion, the human α globin gene driven by its own promoter, the humanξ-globin gene also driven by its own promoter and the optimized β-globingene which includes the porcine introns, poly A and 3' UTR driven by theporcine promoter. The gene order in this construct is LCR ξβ*αα (where *signifies optimized β gene). Constructs 505 or 515 may be used togenerate transgenic pigs expressing human hemoglobin.

Deposit of Microorganisms

The following plasmids were deposited with the American Type CultureCollection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852.

    ______________________________________                                        plasmid       containing    accession no.                                     ______________________________________                                        psaf/pigε (k)                                                                       pigε globin gene                                                                    75371                                               pGem5/Pigβpr (K) pig adult β globin 753727                           gene regulatory region                                                       pPig3'β 3' end of pig 75373                                               β globin gene                                                           pGEM3 β* Δ3' optimized human 75520                                  β globin                                                                pPH1 pig β globin LCR 75518                                              pPH2 pig β globin LCR 75519                                            ______________________________________                                    

Various publications are cited herein which are hereby incorporated byreference in their entirety.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 26                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 889 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - CCCCAGCCCT TTTTCCAGGT CAGCGCAGGG AAAAAACATG TTCTCTGTCC CT -            #GGTTATAC     60                                                                 - - TGTTTAGAAA CATCACCTCC CTCGGCGAAA CTAAAACTTG GGGGTTGCAA TT -            #TATTCCTT    120                                                                 - - GCTTCTTTGT ATTTCGTACC ACATTGAGAG AGCTCTAGGT TTTCATCCGC AG -            #ATTCCCAA    180                                                                 - - ACCTTCGCAG AGGAGCTGTT TCACAGGACC GTGATTCAAG TTTACTCTAC TT -            #TTCCATCA    240                                                                 - - TTTATTTGGT CATATGTTTA AATGAAGAAA GAAAGGAATG AAGATACCTG AA -            #TGAAATGA    300                                                                 - - GTATTTGTTT TCTTACCAGC AGGACTGAAT ACAAATGAAG AGAAGAAAAA TA -            #CGCACATT    360                                                                 - - TAGGACTTGG GCAGAGGTTT TATCCACGCT CTCCTTGTGG TTATTTCCCA TA -            #TTCAGAAG    420                                                                 - - GCGCGGGTGT GGATTCGTCT GTATGGTCCT AAATTGAACC ACAGTGGTCA AA -            #TCCCTCCA    480                                                                 - - CTTTCTGCTC CTTGGATTCT TCGTTTGTGT ACTAAGAAAA TGGGGAGGCA GT -            #CTCTAAGA    540                                                                 - - GATTGCTACA GTGGGACTCA ACTCTAAAAG TTGTACAGAC TTGCTAAGGA GG -            #ATGAAATT    600                                                                 - - AGTAGCACTT TGCACTGTGA GGATGGACCT AGAGCTCCCC AGAGAAGGGC TG -            #AAGGTCTG    660                                                                 - - AAGTTGGTGC CAGGAACGTC TCGAAGACAG GTATACTGTC AACATTCAAG CC -            #TCACCCTG    720                                                                 - - TGGAACCACG CCCTGGCCTG GGCCAATCTG CTCCCAGAAG CAGGGAGGGC AG -            #GAGGCTGG    780                                                                 - - GGGGGCATAA AAGGAAGAGC AGAGCCAGCA GCCACCTACA TTTGCTTCTG AC -            #ACAACCGT    840                                                                 - - GTTCACTAGC AACTGCACAA ACAGACAACA TGGTGCATCT GTCTGCTGA  - #                  889                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 273 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - CCCCAGACAC TCTTGCAGAT TAGTCCAGGC AGAAACAGTT AGATGTCCCC AG -             #TTAACCTC     60                                                                 - - CTATTTGACA CCACTGATTA CCCCATTGAT AGTCACACTT TGGGTTGTAA GT -            #GACTTTTT    120                                                                 - - ATTTATTTGT ATTTTTGACT GCATTAAGAG GTCTCTAGTT TTTTATCTCT TG -            #TTTCCCAA    180                                                                 - - AACCTAATAA GTAACTAATG CACAGAGCAC ATTGATTTGT ATTTATTCTA TT -            #TTTAGACA    240                                                                 - - TAATTTATTA GCATGCATGA GCAAATTAAG AAA       - #                  -      #        273                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 596 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - TTTTTCTTTT CTTACCAGAA GGTTTTAATC CAAATAAGGA GAAGATATGC TT -            #AGAACTGA     60                                                                 - - GGTAGAGTTT TCATCCATTC TGTCCTGTAA GTATTTTGCA TATTCTGGAG AC -            #GCAGGAAG    120                                                                 - - AGATCCATCT ACATATCCCA AAGCTGAATT ATGGTAGACA AAGCTCTTCC AC -            #TTTTAGTG    180                                                                 - - CATCAATTTC TTATTTGTGT AATAAGAAAA TTGGGAAAAC GATCTTCAAT AT -            #GCTTACCA    240                                                                 - - AGCTGTGATT CCAAATATTA CGTAAATACA CTTGCAAAGG AGGATGTTTT TA -            #GTAGCAAT    300                                                                 - - TTGTACTGAT GGTATGGGGC CAAGAGATAT ATCTTAGAGG GAGGGCTGAG GG -            #TTTGAAGT    360                                                                 - - CCAACTCCTA AGCCAGTGCC AGAAGAGCCA AGGACAGGTA CGGCTGTCAT CA -            #CTTAGACC    420                                                                 - - TCACCCTGTG GAGCCACACC CTAGGGTTGG CCAATCTACT CCCAGGAGCA GG -            #GAGGGCAG    480                                                                 - - GAGCCAGGGC TGGGCATAAA AGTCAGGGCA GAGCCATCTA TTGCTTACAT TT -            #GCTTCTGA    540                                                                 - - CACAACTGTG TTCACTAGCA ACCTCAAACA GACACCATGG TGCACCTGAC TC - #CTGA            596                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 477 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - AAAATAAAAG GCAGACAGTC TAAAATAGAA AACCAGTGGT ATNGTNGTTT AT -             #TAATTTGT     60                                                                 - - GCTCATAACT TGAATACTCA TGTCTTTGTG CACAATTATT CTTTCCTTGT AT -            #TGATTAGG    120                                                                 - - TCAAAGTAGA GGAAACCAAC TGTGTCAAAG CAGGAGCTGG ATGCAATCTT GG -            #CAATAAGA    180                                                                 - - ATCTTGCCAG TAGGGTCACG TATGGCTTTT TCCTCCATCT TCAAGGGAAG GA -            #GAGTTTTG    240                                                                 - - GCCAGGACAT AAATGTTACA TGAGGTTCAA AACGTCTCTG GACTGTAAGC CA -            #GGGGAGCA    300                                                                 - - ACCTTCCTTT CCACATACTT TCCTNGCTCG GCTAACTCCC CAATGATAAA CA -            #TGCTTCTC    360                                                                 - - TTTATACAAT AGACATTCCA CATGTTATAG TTAAGAGCTT CCAGCCTGGG AG -            #TCATTCTG    420                                                                 - - TATCTTTCAG GTGACTTTGA GACACTTTTC CTATCAGTTA ATTTACTTTT GA - #TCCTC           477                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 403 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - GTTTTTTACA CTGGAATTTA TAACTAGAGC ACTCATGTTT ATGTAAGCAA TT -             #AATTGTTT     60                                                                 - - CATCAGTCAG GTAAAAGTAA AGAAAAACTG TGCCAAGGCA GGTAGCCTAA TG -            #CAATATGC    120                                                                 - - CACTAAAGTA AACATTATTC CATAGGTGTC AGATATGGCT TATTCATCCA TC -            #TTCATGGG    180                                                                 - - AAGGATGGCC TTGGCCTGGA CATCAGTGTT ATGTGAGGTT CAAAACACCT CT -            #AGGCTATA    240                                                                 - - AGGCAACAGA GCTCCTTTTT TTTTTTTCTG TGCTTTCCTG GCTGTCCAAA TC -            #TCTAATGA    300                                                                 - - TAAGCATACT TCTATTCAAT GAGAATATTC TGTAAGATTA TAGTTAAGAA TT -            #GTGGGAGC    360                                                                 - - CATTCCGTCT CTTATAGTTA AATTTGAGCT TCTTTTATGA TCA    - #                      - #403                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 998 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - GATCTCACGT ATATACGCAC CTAAAAAGTT GAATACATAG AGCTGCGAGT AG -             #ACGGTGGC     60                                                                 - - TGCAGGGATG GGGAAAGTGG GAGAANCCAC TCAGATCTGG GTCAAGGGCA CA -            #CGTCTTCA    120                                                                 - - NNNATCTTTC AGTGACGTNA AGACGTGGAG GTCTAATGGC TTACGGACTG TA -            #GTAATGAC    180                                                                 - - GCAGCACCGA ACGCTNGGAC ATGTGCTAAG ATTTCGGGTG TTCTCATCAC AC -            #CCCCAAAG    240                                                                 - - TGGCAACTGT GAGGAAAGAC AGTTAAGTAA CCTGACTGAG GAGCCGTTTC CC -            #TGTGTCTG    300                                                                 - - TGTCATACAC CTCGCATTAC ACCTCGCATT ACACGAGTTG CATCAAAAAA GA -            #AAGTATTC    360                                                                 - - AAAATAGCTA TATTTCTAAT CATCCTTTGG AGTTGAGATG TGAGCCGAAG AG -            #TTACATGT    420                                                                 - - ACATGCTTGA CATTTGAACT CGAAATAATA TTTAGGGAGC ATGTATGATT TC -            #TCTATCCC    480                                                                 - - TTTACACAAT AAACTAAAAT AATTCTCATG ATTTACCCTA TGAGCTCCCC TC -            #CAAGGCTA    540                                                                 - - CGTGGCTCTG TCTCACGGTG TCATCCGTTG TAGCCTGTTC TGCCCGCCCG GC -            #CTTAAGGC    600                                                                 - - AGGTGGAGGA CAGGTATATC CTTGCCTTAT GGAAAATCCA CTGCGTCTTT CA -            #AGGCCCAG    660                                                                 - - TTTATTGTTC CTTTGGTTCC ATGAGACTTT TGGTAGCTCA CTCCCTCCCT AA -            #AAGGAACC    720                                                                 - - CAGACTGAGG GTGGTATTTC CCTCCCATAT ATTTCTCTTT TAAGTGTGGA AA -            #AGGTATTC    780                                                                 - - TAATAGTACA TATAATTATC GACTGGTTTG TTGTTGTTGT TCTTTTTTGG CC -            #GTACCTGC    840                                                                 - - AGCATATGAA CGTTCCTGGG CCAGGGACAG AATCCAAGCC AGAGCTGCGC CC -            #TCCCCCAG    900                                                                 - - AGCTACGGCA GTGCTGGATT CTTAACCGCT GTGCTGGGCC CGGATGTGAA CC -            #CGCAACGC    960                                                                 - - TACAGAGACT GAGCCGGATC GTTAACCGCT GCACTGCG      - #                      - #    998                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 166 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - AAGAAATACC TCCGAATAAC TGTACCTCCA ATTATTCTTT AAGGTAGCAT GC -             #AACTGTAA     60                                                                 - - TAGTTGCATG TATATATTTA TCATAATACT GTAACAGAAA ACACTTACTG AA -            #TATATACT    120                                                                 - - GTGTCCCTAG TTCTTTACAC AATAAACTAA TCTCATCCTC ATAATT   - #                    166                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 234 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - GACTAAGTCA CTCTGTCTCA CTGTGTCTTA GCCAGTTCCT TACAGCTTGC CC -             #TGATGGGA     60                                                                 - - GATAGAGAAT GGGTATCCTC CAACAAAAAA ATAAATTTTC ATTTCTCAAG GT -            #CCAACTTA    120                                                                 - - TGTTTTCTTA ATTTTTAAAA AAATCTTGAC CATTCTCCAC TCTCTAAAAT AA -            #TCCACAGT    180                                                                 - - GAGAGAAACA TTCTTTTCCC CCATCCCATA AATACCTCTA TTAAATATGG AA - #AA              234                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - CCTCTAAGAC TAAGTCACTC TGTCTCACTG TGTC       - #                  -      #        34                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 282 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - CCCCAAGTCC TGGTCGAGGG CCTGTCCATG GCGATTAAAT CACCCCAAGA AA -            #GTCCCCGT     60                                                                 - - CCTTCTCTGC GCTTCAGCCC CCTCTTCTGT AAAGGGCCTG CAAAGGGCCC TC -            #TGCCGCCG    120                                                                 - - GAGAATTTCT CCTGCTGAAA CACACAGGCT CCCTCAGCTC AACCGGGACT GT -            #CGCTACAT    180                                                                 - - CTATCACTTC TTCGCCTGCA CGACATCTGG GGTCTCTCAT CAGGGAGGGC CT -            #TCTCTTCT    240                                                                 - - AAACCAAGCC CACCGGGCCC TGGGAGCGTG GGAGCAGAGA GG    - #                      - # 282                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - TCATACTGAG AAAGTCCCCA CCCTTCTCTG AGCCTCAG      - #                      - #     38                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - ATGGTGCACC TGACTCCTGA GGAGAAGTCT GCCGTTACTG CCCTGTGGGG CA -             #AGGTGAAC     60                                                                 - - GTGGATGAAG TTGGTGGTGA GGCCCTGGGC AGG       - #                  -      #         93                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 222 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - CTGCTGGTGG TCTACCCTTG GACCCAGAGG TTCTTTGAGT CCTTTGGGGA TC -            #TGTCCACT     60                                                                 - - CCTGATGCTG TTATGGGCAA CCCTAAGGTG AAGGCTCATG GCAAGAAAGT GC -            #TCGGTGCC    120                                                                 - - TTTAGTGATG GCCTGGCTCA CCTGGACAAC CTCAAGGGCA CCTTTGCCAC AC -            #TGAGTGAG    180                                                                 - - CTGCACTGTG ACAAGCTGCA CGTGGATCCT GAGAACTTCA GG    - #                      - # 222                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 129 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - CTCCTGGGCA ACGTGCTGGT CTGTGTGCTG GCCCATCACT TTGGCAAAGA AT -             #TCACCCCA     60                                                                 - - CCAGTGCAGG CTGCCTATCA GAAAGTGGTG GCTGGTGTGG CTAATGCCCT GG -            #CCCACAAG    120                                                                 - - TATCACTAA                - #                  - #                      - #        129                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - ATGGTGCATC TGTCTGCTGA GGAGAAGGAG GCCGTCCTCG GCCTGTGGGG CA -             #AAGTGAAT     60                                                                 - - GTGGACGAAG TTGGTGGTGA GGCCCTGGGC AGG       - #                  -      #         93                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 222 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - CTGCTGGTTG TCTACCCCTG GACTCAGAGG TTCTTCGAGT CCTTTGGGGA CC -            #TGTCCAAT     60                                                                 - - GCCGATGCCG TCATGGGCAA TCCCAAGGTG AAGGCCCACG GCAAGAAGGT GC -            #TCCAGTCC    120                                                                 - - TTCAGTGACG GCCTGAAACA TCTCGACAAC CTCAAGGGCA CCTTTGCTAA GC -            #TGAGCGAG    180                                                                 - - TCGCACTGTG ACCAGCTGCA CGTGGATCCT GAGAACTTCA GG    - #                      - # 222                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 129 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - CTCCTGGGCA ACGTGATAGT GGTTGTTCTG GCTCGCCGCC TTGGCCATGA CT -             #TCAACCCG     60                                                                 - - AATGTGCAGG CTGCTTTTCA GAAGGTGGTG GCTGGTGTTG CTAATGCCCT GG -            #CCCACAAG    120                                                                 - - TACCACTAA                - #                  - #                      - #        129                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 95 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 3..95                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - CC ATG GTG CAT CTG ACT CCT GAG GAG AAG TCT - # GCC GTC ACT GCC CTG            47                                                                          Met Val His Leu Thr Pro Glu Glu Lys - #Ser Ala Val Thr Ala Leu                  1             - #  5                - #  10                - #  15        - - TGG GGC AAA GTG AAT GTG GAC GAA GTT GGT GG - #T GAG GCC CTG GGC AGG           95                                                                       Trp Gly Lys Val Asn Val Asp Glu Val Gly Gl - #y Glu Ala Leu Gly Arg                            20 - #                 25 - #                 30              - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - Met Val His Leu Thr Pro Glu Glu Lys Ser Al - #a Val Thr Ala Leu Trp        1               5 - #                 10 - #                 15              - - Gly Lys Val Asn Val Asp Glu Val Gly Gly Gl - #u Ala Leu Gly Arg                       20     - #             25     - #             30                  - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 222 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..222                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - CTG CTG GTT GTC TAC CCC TGG ACT CAG AGG TT - #C TTC GAG TCC TTT GGG           48                                                                       Leu Leu Val Val Tyr Pro Trp Thr Gln Arg Ph - #e Phe Glu Ser Phe Gly             1               5 - #                 10 - #                 15              - - GAC CTG TCC ACT CCT GAT GCC GTC ATG GGC AA - #T CCC AAG GTG AAG GCC           96                                                                       Asp Leu Ser Thr Pro Asp Ala Val Met Gly As - #n Pro Lys Val Lys Ala                        20     - #             25     - #             30                  - - CAC GGC AAG AAG GTG CTC GGT GCC TTC AGT GA - #C GGC CTG GCT CAT CTC          144                                                                       His Gly Lys Lys Val Leu Gly Ala Phe Ser As - #p Gly Leu Ala His Leu                    35         - #         40         - #         45                      - - GAC AAC CTC AAG GGC ACC TTT GCT ACA CTG AG - #C GAG CTG CAC TGT GAC          192                                                                       Asp Asn Leu Lys Gly Thr Phe Ala Thr Leu Se - #r Glu Leu His Cys Asp                50             - #     55             - #     60                          - - AAG CTG CAC GTG GAT CCT GAG AAC TTC AGG  - #                  - #              222                                                                     Lys Leu His Val Asp Pro Glu Asn Phe Arg                                        65                 - # 70                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 74 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - Leu Leu Val Val Tyr Pro Trp Thr Gln Arg Ph - #e Phe Glu Ser Phe Gly        1               5 - #                 10 - #                 15              - - Asp Leu Ser Thr Pro Asp Ala Val Met Gly As - #n Pro Lys Val Lys Ala                   20     - #             25     - #             30                  - - His Gly Lys Lys Val Leu Gly Ala Phe Ser As - #p Gly Leu Ala His Leu               35         - #         40         - #         45                      - - Asp Asn Leu Lys Gly Thr Phe Ala Thr Leu Se - #r Glu Leu His Cys Asp           50             - #     55             - #     60                          - - Lys Leu His Val Asp Pro Glu Asn Phe Arg                                   65                 - # 70                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 129 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..129                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - CTC CTG GGC AAC GTG CTG GTG TGT GTT CTG GC - #T CAT CAC TTT GGC AAA           48                                                                       Leu Leu Gly Asn Val Leu Val Cys Val Leu Al - #a His His Phe Gly Lys             1               5 - #                 10 - #                 15              - - GAA TTC ACC CCG CCG GTG CAG GCT GCT TAT CA - #G AAG GTG GTG GCT GGT           96                                                                       Glu Phe Thr Pro Pro Val Gln Ala Ala Tyr Gl - #n Lys Val Val Ala Gly                        20     - #             25     - #             30                  - - GTT GCT AAT GCC CTG GCC CAC AAG TAC CAC TA - #A                  -      #        129                                                                    Val Ala Asn Ala Leu Ala His Lys Tyr His                                                35         - #         40                                             - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - Leu Leu Gly Asn Val Leu Val Cys Val Leu Al - #a His His Phe Gly        Lys                                                                               1               5 - #                 10 - #                 15             - - Glu Phe Thr Pro Pro Val Gln Ala Ala Tyr Gl - #n Lys Val Val Ala Gly                   20     - #             25     - #             30                  - - Val Ala Asn Ala Leu Ala His Lys Tyr His                                           35         - #         40                                             - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - Met Val His Leu Thr Pro Glu Glu Lys Ser Al - #a Val Thr Ala Leu Trp        1               5 - #                 10 - #                 15              - - Gly Lys Val Asn Val Asp Glu Val Gly Gly Gl - #u Ala Leu Gly Arg                       20     - #             25     - #             30                  - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 74 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - Leu Leu Val Val Tyr Pro Trp Thr Gln Arg Ph - #e Phe Glu Ser Phe Gly        1               5 - #                 10 - #                 15              - - Asp Leu Ser Thr Pro Asp Ala Val Met Gly As - #n Pro Lys Val Lys Ala                   20     - #             25     - #             30                  - - His Gly Lys Lys Val Leu Gly Ala Phe Ser As - #p Gly Leu Ala His Leu               35         - #         40         - #         45                      - - Asp Asn Leu Lys Gly Thr Phe Ala Thr Leu Se - #r Glu Leu His Cys Asp           50             - #     55             - #     60                          - - Lys Leu His Val Asp Pro Glu Asn Phe Arg                                   65                 - # 70                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - Leu Leu Gly Asn Val Leu Val Cys Val Leu Al - #a His His Phe Gly Lys        1               5 - #                 10 - #                 15              - - Glu Phe Thr Pro Pro Val Gln Ala Ala Tyr Gl - #n Lys Val Val Ala Gly                   20     - #             25     - #             30                  - - Val Ala Asn Ala Leu Ala His Lys Tyr His                                           35         - #         40                                           __________________________________________________________________________

What is claimed is:
 1. A purified and isolated nucleic acid comprisingthe sequence as set forth in SEQ. ID NO: 1.